Method for inducing NK cells from placenta-derived hematopoietic stem cells

Abstract

The invention provides a method for inducing NK (Natural Killer) cells by using placenta-derived hematopoietic stem cells, which comprises the following steps: selecting a full-month placenta sample of a healthy pregnant woman without infectious diseases, and separating mononuclear cells from the placenta sample; adopting a Meitenyi magnetic bead sorting method to sort CD34 + cells; and inducing NK culture by using CD34 + cells. According to the preparation method for inducing the placenta-derived hematopoietic stem cells into the NK through the culture medium composed of the lipoic acid, the nicotinamide and the interleukin, the preparation period can be shortened to 4 weeks, the cell amplification multiple can reach more than 4000 times, the purity of the NK is larger than 95%, and the NK can be clinically and massively used.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell induction, and in particular to a method for inducing NK cells with placenta-derived hematopoietic stem cells. Background Art

[0002] Natural killer cells are a type of lymphocytes that develop from pluripotent stem cells in the bone marrow. They are usually divided into two categories based on the expression levels of two surface markers, CD56 and CD16: CD56brightCD16+ / - and CD56dimCD16brght. CD56brightNK cells are mainly found in lymph nodes and other lymphoid tissues, have strong immunoregulatory functions, and regulate immune responses by secreting cytokines. In contrast, CD56dimNK cells mainly circulate in the peripheral blood, directly kill target cells by releasing perforin, etc., and have strong cytotoxic effects. Natural killer cell adoptive therapy was first used in hematopoietic stem cell transplantation to enhance the graft-versus-leukemia effect, reduce graft-versus-host disease, and prolong the survival time of hematopoietic stem cell transplantation. Today, natural killer cells are most commonly allogeneic cells, including peripheral blood, umbilical cord blood, placenta, CD34+ hematopoietic stem cells, and pluripotent stem cells.

[0003] The results of the clinical trial NCT05020678 of gene-modified natural killer cells for the treatment of non-Hodgkin's lymphoma or chronic lymphocytic leukemia showed that the objective response rate of patients was 72.73%, of which 7 cases were completely relieved and no serious adverse reactions occurred. Among these reported NK preparation methods, most use mononuclear cells from peripheral blood, umbilical cord blood, and other sources as samples for amplification and culture of NK. The stability of the source and its quality are important factors affecting the expansion of the number of NK cells. However, most use hematopoietic stem cells from umbilical cord blood to induce NK, but the content of umbilical cord blood is affected by the time of cord cutting, and the hematopoietic stem cells collected from umbilical cord blood often cannot meet the needs of mass production of NK. Therefore, the placenta source has become the best choice for the development of allogeneic gene therapy products.

[0004] The placenta is an important hematopoietic organ in the early stage of fetal development. It is rich in hematopoietic progenitor cells. After in vitro expansion and induction into adoptive immunotherapy cells such as NK, it can be used for clinical auxiliary transfusion therapy. For example, Celularity's CYNK-001 has completed phase I clinical trials with significant efficacy. However, in vitro directed differentiation is difficult, it is difficult to obtain high-purity NK, and the cycle is long. How to shorten the preparation cycle, reduce production costs and meet clinical needs has become a problem. Summary of the invention

[0005] The purpose of the present invention is to propose a method for inducing NK cells from placenta-derived hematopoietic stem cells, aiming to solve the problems of difficulty in obtaining high-purity NK and long cycle in traditional NK cell differentiation technology.

[0006] In a first aspect, the present invention provides a method for inducing NK cells with placenta-derived hematopoietic stem cells, the method comprising:

[0007] Select full-term placenta samples from healthy pregnant women without infectious diseases, and isolate mononuclear cells from the placenta samples;

[0008] CD34+ cells were sorted using Miltenyi magnetic bead sorting method;

[0009] NK cell culture was induced using CD34+ cells.

[0010] In some embodiments, the step of selecting a full-term placenta sample from a healthy pregnant woman without infectious diseases and isolating mononuclear cells from the placenta sample comprises:

[0011] The placenta was taken out and laid flat on a stainless steel plate. The residual blood clots on the surface were washed with physiological saline. After cleaning, the placenta was turned over to expose the parietal decidua surface.

[0012] The amniotic membrane was peeled off, the parietal decidua tissue was cut off, anticoagulant buffer was added, the placental tissue was soaked, the lobular tissue was cut off, the lobular tissue was recovered and fully shredded, and filtered with a 200-mesh filter;

[0013] Add anticoagulant buffer to rinse the placental tissue, recover the eluate into a centrifuge tube, aspirate the supernatant, and add physiological saline to resuspend;

[0014] Use density gradient centrifugation to separate mononuclear cells, grind the tube wall and slowly add it to the upper layer of lymphocyte separation solution in the separation solution tube;

[0015] After centrifugation, the buffy coat cells were collected, washed with physiological saline, centrifuged at 1800 rpm for 10 min, the excess supernatant was discarded, physiological saline was added to resuspend the cells, and the cells were counted.

[0016] In some embodiments, the step of sorting CD34+ cells using Miltenyi magnetic bead sorting method comprises:

[0017] Magnetic bead labeling: add 300ul sorting buffer to resuspend cells per 1*10^8 total cells, add 100ul FcR Blocking Reagent per 1*10^8 total cells, add 100ul CD34 MicroBeads per 1*10^8 total cells, mix well and incubate at 4℃ for 30min;

[0018] For every 1*10^8 total cells, add 10 ml of sorting buffer to wash, centrifuge at 300 g for 10 min, remove the supernatant, and add 500 ul of sorting buffer to resuspend the cells for column sorting;

[0019] Wash the sorting column, load the sample for sorting, the effluent is CD34- cells, remove the sorting column and add sorting buffer to elute the CD34+ cells, centrifuge at 300g for 10 minutes, remove the supernatant and add 10ml of the pre-configured first induction culture medium, take samples and count them for later use.

[0020] In some embodiments, the step of inducing NK culture using CD34+ cells comprises:

[0021] After obtaining the counts, CD34+ cells were seeded in a 6-well plate at a density of 0.5*10^5 cells / ml, recorded as Day 0;

[0022] Day 4 later, rewarm the first induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10 minutes, discard the supernatant, and add fresh first induction medium at a density of 0.5*10^5-1*10^5 cells / ml;

[0023] On day 7, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh second induction medium at a density of 1*10^5 cells / ml;

[0024] On Day 9-13, observe and change the medium every two days, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh second induction medium at a density of 5*10^5 cells / ml;

[0025] On Day 14, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, add NK expansion reagent A, mix well, and place in the incubator for static culture.

[0026] On Day 17, take a small amount of cell suspension from the rewarmed amplification medium for counting and add the rewarmed amplification medium at a density of 1*10^6;

[0027] On Day 19, take a small amount of cell suspension from the rewarmed expansion medium for counting and add the rewarmed expansion medium at a density of 1*10^6;

[0028] On Day 21, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, add NK expansion reagent B, mix well and place in the incubator for static culture.

[0029] On Day 22-28, observe the fluid replenishment every day, add rewarmed expansion medium at a density of 1*10^6, and harvest the cells on Day 28.

[0030] In some embodiments, the first induction medium comprises 450 ml SCGM Media, 50 ml human AB serum, 5 ug IL-3, 10 ug IL-7, 5 ug IL-15, 10 ug SCF, 5 ug Flt3L, 5 ug IL-21, and 13.5 mg α-lipoic acid.

[0031] In some embodiments, the second induction medium comprises 450 ml SCGM Media, 50 ml human AB serum, 10 ug IL-7, 5 ug IL-15, 10 ug SCF, 5 ug Flt3L, 5 ug IL-21, 10 mM nicotinamide, and 13.5 mg α-lipoic acid.

[0032] In some embodiments, the expansion medium comprises 450 ml KBM 581 lymphocyte serum-free medium, 50 ml human AB serum, 200 IU / ml IL-2, and 10 mM nicotinamide.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The present invention provides a preparation method for inducing placenta-derived hematopoietic stem cells into NK using a culture medium composed of lipoic acid, nicotinamide and interleukin. The preparation cycle can be shortened to 4 weeks, the cell expansion multiple can reach more than 4000 times, the NK purity is greater than 95%, and the NK can be used in large quantities in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 (a) is a graph showing the CD34+ ratio before magnetic bead separation in Example 2. Figure 1 (b) is a flow cytometry analysis of the CD34+ ratio after sorting in Example 2. Figure 1 (c) is a CD34-ratio diagram detected by flow cytometry after sorting in Example 2;

[0036] Figure 2 (a) is a graph showing the detection of erythroid colony-forming units of placenta-derived CD34+ hematopoietic stem cells in Example 2; Figure 2 (b) is a detection diagram of placenta-derived CD34+ hematopoietic stem cell granulocyte / macrophage colony-forming units in Example 2; Figure 2(c) is a diagram showing a mixed colony detection of placenta-derived CD34+ hematopoietic stem cells formed by granulocytes, erythrocytes, macrophages, and megakaryocytes in Example 2;

[0037] Figure 3 (a) is a schematic diagram of the cell morphology characteristics of Example 4 after culturing with the first culture medium on day 0. The extracted hematopoietic stem cells are oval in shape with clear and bright edges; Figure 3 (b) is a schematic diagram of the cell morphology after culturing in the first culture medium for 7 days in Example 4. Under a microscope, many cells are observed and some cells aggregate;

[0038] Figure 4 This is a schematic diagram of the cell morphology characteristics after culturing for 14 days using the second culture medium in Example 4. Under a microscope, the cells are densely packed, clustered, and the edges of individual cells are clear;

[0039] Figure 5 This is a schematic diagram of the cell morphology characteristics after culturing for 28 days using the third culture medium in Example 4. Under a microscope, the cell density is high, there are obvious clusters, the individual cells are clear and bright, and some cells are irregular;

[0040] Figure 6 This is the flow cytometry graph of placenta-derived CD34+ cells inducing NK cells on day 28 in Example 4. The CD3-CD56+ ratio is 96.82%, and the CD56+CD16+ ratio is 71.1%;

[0041] Figure 7 This is the flow cytometry chart of comparative example 1, in which placenta-derived CD34+ cells induced NK cells on day 28, with a CD3-CD56+ ratio of 53.74% and a CD56+CD16+ ratio of 37.89%;

[0042] Figure 8 This is the flow cytometry chart of comparative example 2, in which placenta-derived CD34+ cells induced NK cells on day 28, with a CD3-CD56+ ratio of 58.82% and a CD56+CD16+ ratio of 40.81%;

[0043] Fig. 9 The statistical graph of NK amplification induced by placenta-derived CD34+ cells in Example 4, Comparative Example 1, and Comparative Example 2 shows that Example 4 has the highest amplification multiple of 4420 times, while Comparative Example 1 and Comparative Example 2 are 2433 times and 2843 times, respectively;

[0044] Fig.10 The figures are for Example 4, Comparative Example 1, and Comparative Example 2, in which the NK cells induced by placenta-derived CD34+ cells kill K562 cells. The killing rate of Example 4 is 89.13% when the effector-target ratio is 20:1, while that of Comparative Example 1 and Comparative Example 2 are 62.2% and 59.07%, respectively. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0046] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0047] Example 1

[0048] Mononuclear cell extraction

[0049] 1. Select a full-term placenta sample from a healthy pregnant woman without infectious diseases. The mother did not have heavy bleeding during the birth of the baby. The placenta should not be refrigerated for more than 24 hours;

[0050] 2. Take out the placenta and lay it flat on a stainless steel plate. Use physiological saline to clean the remaining blood clots on the surface. After cleaning, turn the placenta over to expose the parietal decidua surface;

[0051] 3. Use forceps to peel off the amniotic membrane, use surgical scissors to cut off the parietal decidua tissue, add anticoagulant buffer, and soak the placental tissue. Use elbow scissors to cut off the lobular tissue. Recover the lobular tissue and cut it into pieces, and filter it with a 200-mesh filter.

[0052] 4. Add anticoagulant buffer to rinse the placenta tissue and recover the eluate into a centrifuge tube. Centrifuge at 2000rpm for 10min (9 for ascending and 6 for descending), aspirate the supernatant, and retain a total volume of 20ml in each centrifuge bottle. Add physiological saline to resuspend;

[0053] 5. Use density gradient centrifugation to separate mononuclear cells. Grind the tube wall and slowly add it to the upper layer of lymphocyte separation solution in the separation solution tube, centrifuge at 800×g for 20 minutes (7 for ascending and 0 for descending);

[0054] 6. After centrifugation, collect the buffy coat cells, wash with saline, centrifuge at 1800 rpm for 10 min, discard the excess supernatant, add saline to resuspend the cells, and count.

[0055] Example 2

[0056] CD34+ cell sorting

[0057] CD34+ cells were sorted using Miltenyi magnetic bead sorting method, the steps are as follows;

[0058] 1. Magnetic bead labeling: add 300ul sorting buffer to resuspend cells for every 1*10^8 total cells, add 100ul FcR Blocking Reagent for every 1*10^8 total cells, add 100ul CD34 MicroBeads for every 1*10^8 total cells, mix well and incubate at 4℃ for 30min.

[0059] 2. Add 10 ml of sorting buffer to wash every 1*10^8 total cells, centrifuge at 300g for 10 minutes, remove the supernatant, add 500ul of sorting buffer to resuspend the cells for column sorting;

[0060] 3. Wash the sorting column, load the sample for sorting, the effluent is CD34- cells, remove the sorting column and add sorting buffer to elute the CD34+ cells, centrifuge at 300g for 10 minutes, remove the supernatant and add 10ml of the first induction culture medium, take samples and count, inoculate the hematopoietic stem cell colony experiment according to the counting results, and the remaining cells are reserved, as shown in the following Table 1, which is a statistical table of the number of hematopoietic stem cell colonies:.

[0061] Table 1 Statistics of the number of hematopoietic stem cell colonies

[0062] Number of colonies D7 D14 CD34+ 107 110 CD34- 0 0

[0063] Example 3

[0064] Medium preparation

[0065] 1. The first induction medium is prepared as shown in Table 2:

[0066] Table 2

[0067] Product Name Dosage SCGMMedia 450ml Human AB serum 50ml IL-3 5ug IL-7 10ug IL-15 5ug SCF 10ug F3L 5ug IL-21 5ug Alpha-lipoic acid (ALA) 13.5mg

[0068] 2. The second induction medium is prepared as shown in Table 3:

[0069] Table 3

[0070] Product Name Dosage SCGMMedia 450ml Human AB serum 50ml IL-7 10ug IL-15 5ug SCF 10ug F3L 5ug IL-21 5ug Niacinamide 10mM Alpha-lipoic acid (ALA) 13.5mg

[0071] 3. Preparation of expansion medium, as shown in Table 4:

[0072] Table 4

[0073]

[0074] Example 4

[0075] CD34+ cell-induced NK cell culture

[0076] 1. After obtaining the count, inoculate the cells in a 6-well plate at a density of 0.5*10^5Cells / ml, which is recorded as Day 0; 2. After Day 4, rewarm the first induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh first induction medium at a density of 0.5-1*10^5Cells / ml;

[0077] 3. On Day 7, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10 minutes, discard the supernatant, and add fresh second induction medium at a density of 1*10^5 cells / ml;

[0078] 4. On Day 9-13, observe and change the medium every two days, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh second induction medium at a density of 5*10^5 cells / ml;

[0079] 5. On Day 14, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, and add NK expansion reagent A (Shouning Bio, NK expansion kit, product number RP03030-B), mix well, and place in the incubator for static culture;

[0080] 6. On Day 17, take a small amount of cell suspension from the rewarmed expansion medium for counting and add the rewarmed expansion medium at a density of 1*10^6;

[0081] 7. On Day 19, take a small amount of cell suspension from the rewarmed expansion medium for counting and add the rewarmed expansion medium at a density of 1*10^6;

[0082] 8. On Day 21, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, and add NK expansion reagent B (Shouning Bio, NK expansion kit, catalog number RP03030-B). Mix well and place in the incubator for static culture.

[0083] 9. On Day 22-28, observe the rehydration every day and add rewarmed expansion medium at a density of 1*10^6. Harvest the cells on Day 28.

[0084] The cells harvested in Example 4 were tested for purity, and the purity of the induced NK cells was 96.82%, and the cell expansion multiple was 4420 times.

[0085] Comparative Example 1

[0086] This comparative example is basically the same as Example 4, except that the second induction medium is changed to the first induction medium, and the purity of the induced NK cells is 53.74%, and the cell expansion multiple is 2433 times.

[0087] Comparative Example 2

[0088] This comparative example is basically the same as Example 4, except that the first induction medium is changed to the second induction medium, and the purity of the induced NK cells is 58.82%, and the cell expansion multiple is 2843 times.

[0089] Depend on Figure 1 It can be seen that the CD34 ratio before sorting and enrichment (before sorting) was 1.23%. After enrichment with Miltenyi CD34 magnetic beads, the CD34 ratio increased to 52.67%, indicating that the use of Miltenyi CD34 magnetic beads can enrich CD34+ cells from placenta. In addition, the extracted hematopoietic stem cells have the ability to differentiate into a variety of blood cells (as shown in the figure, Figure 2 , Table 3). When the induction medium of the present invention is used for induction culture, obvious cell clusters can be observed under the microscope, and the individual cells are oval in shape with clear and bright edges, indicating that the induction medium is conducive to the induction activation and expansion of hematopoietic stem cells into NK cells (as shown in the figure, Figure 4-6 ); When the induction culture was continued for 28 days, the CD3-CD56+ ratio of Example 4 was 96.82%, the CD3-CD56+ ratio of Comparative Example 1 was 53.74%, and the CD3-CD56+ ratio of Comparative Example 2 was 58.82%, indicating that the NK purity of the induction culture medium using Example 4 was higher (as shown in the figure, Figure 7-9 ). According to the amplification statistics, the amplification multiple of Example 4 is better than that of Comparative Example 1 and Comparative Example 2 (as shown in the figure, Fig. 9 In addition, the results of the K562 killing ability graph show that the NK cells using the induced expansion medium of Example 4 have a stronger ability to kill K562 (as shown in the figure, Fig.10 ).

[0090] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for inducing NK cells from placenta-derived hematopoietic stem cells, characterized in that: The method comprises: Select full-term placenta samples from healthy pregnant women without infectious diseases, and isolate mononuclear cells from the placenta samples; CD34+ cells were sorted using Miltenyi magnetic bead sorting method; NK cell culture was induced using CD34+ cells.

2. The method for inducing NK cells from placenta-derived hematopoietic stem cells according to claim 1, characterized in that: The steps of selecting a full-term placenta sample from a healthy pregnant woman without infectious diseases and isolating mononuclear cells from the placenta sample include: The placenta was taken out and laid flat on a stainless steel plate. The residual blood clots on the surface were washed with physiological saline. After cleaning, the placenta was turned over to expose the parietal decidua surface. The amniotic membrane was peeled off, the parietal decidua tissue was cut off, anticoagulant buffer was added, the placental tissue was soaked, the lobular tissue was cut off, the lobular tissue was recovered and fully shredded, and filtered with a 200-mesh filter; Add anticoagulant buffer to rinse the placental tissue, recover the eluate into a centrifuge tube, aspirate the supernatant, and add physiological saline to resuspend; Use density gradient centrifugation to separate mononuclear cells, grind the tube wall and slowly add it to the upper layer of lymphocyte separation solution in the separation solution tube; After centrifugation, the buffy coat cells were collected, washed with physiological saline, centrifuged at 1800 rpm for 10 min, the excess supernatant was discarded, physiological saline was added to resuspend the cells, and the cells were counted.

3. The method for inducing NK cells with placenta-derived hematopoietic stem cells according to claim 2, characterized in that: The steps of sorting CD34+ cells using Miltenyi magnetic bead sorting method include: Magnetic bead labeling: add 300ul sorting buffer to resuspend cells per 1*10^8 total cells, add 100ul FcR Blocking Reagent per 1*10^8 total cells, add 100ul CD34 MicroBeads per 1*10^8 total cells, mix well and incubate at 4℃ for 30min; For every 1*10^8 total cells, add 10 ml of sorting buffer to wash, centrifuge at 300 g for 10 min, remove the supernatant, and add 500 ul of sorting buffer to resuspend the cells for column sorting; Wash the sorting column, load the sample for sorting, the effluent is CD34- cells, remove the sorting column and add sorting buffer to elute the CD34+ cells, centrifuge at 300g for 10 minutes, remove the supernatant and add 10ml of the pre-configured first induction culture medium, take samples and count them for later use.

4. The method for inducing NK cells with placenta-derived hematopoietic stem cells according to claim 3, characterized in that: The step of inducing NK culture using CD34+ cells comprises: After obtaining the counts, CD34+ cells were seeded in a 6-well plate at a density of 0.5*10^5 cells / ml, recorded as Day 0; Day 4 later, rewarm the first induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10 minutes, discard the supernatant, and add fresh first induction medium at a density of 0.5*10^5-1*10^5 cells / ml; On day 7, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh second induction medium at a density of 1*10^5 cells / ml; On Day 9-13, observe and change the medium every two days, rewarm the second induction medium, remove the cells and take a small amount of cell suspension for counting, centrifuge at 300g for 10min, discard the supernatant, and add fresh second induction medium at a density of 5*10^5 cells / ml; On Day 14, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, add NK expansion reagent A, mix well, and place in the incubator for static culture. On Day 17, take a small amount of cell suspension from the rewarmed expansion medium for counting and add the rewarmed expansion medium at a density of 1*10^6; On Day 19, take a small amount of cell suspension from the rewarmed expansion medium for counting and add the rewarmed expansion medium at a density of 1*10^6; On Day 21, remove all cells and replace the medium. Add the rewarmed expansion medium at a density of 1*10^6, add NK expansion reagent B, mix well and place in the incubator for static culture. On Day 22-28, observe the fluid replenishment every day, add rewarmed expansion medium at a density of 1*10^6, and harvest the cells on Day 28.

5. The method for inducing NK cells with placenta-derived hematopoietic stem cells according to claim 4, characterized in that: The first induction medium includes 450 ml SCGM Media, 50 ml human AB serum, 5 ug IL-3, 10 ug IL-7, 5 ug IL-15, 10 ug SCF, 5 ug Flt3L, 5 ug IL-21, and 13.5 mg α-lipoic acid.

6. The method for inducing NK cells with placenta-derived hematopoietic stem cells according to claim 4, characterized in that: The second induction medium includes 450 ml SCGM Media, 50 ml human AB serum, 10 ug IL-7, 5 ug IL-15, 10 ug SCF, 5 ug Flt3L, 5 ug IL-21, 10 mM nicotinamide, and 13.5 mg α-lipoic acid.

7. The method for inducing NK cells from placenta-derived hematopoietic stem cells according to claim 4, characterized in that: The expansion medium includes 450 ml KBM 581 lymphocyte serum-free medium, 50 ml human AB serum, 200 IU / ml IL-2, and 10 mM nicotinamide.

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