Method for producing CD34-positive hematopoietic stem cells by using secreted proteins from op9 cells

Abstract

The present invention relates to a method for producing CD34-positive hematopoietic stem cells by using secreted proteins from OP9 cells, and the method for producing CD34-positive hematopoietic stem cells, according to the present invention, is characterized by: using OP9 cell culture medium to overcome the limitations of complex OP9 cell co-culture techniques; forming aggregates of human pluripotent stem cells to enable mass production of hematopoietic stem cells; and adding stage-specific growth factors at each differentiation stage to improve culture efficiency, thereby maximizing the production efficiency of CD34-positive cells, and thus hematopoietic stem cells produced by the method of the present invention can be transplanted into humanized mice to obtain a model having an immune system similar to that of humans.

Description

Method for producing CD34-positive hematopoietic stem cells using secreted proteins of OP9 cells

[0001] The present invention relates to a method for producing CD34 positive hematopoietic stem cells using a secretory protein of OP9 cells.

[0002] Humanized mice are used to study human-specific diseases such as HIV / AIDS, cancer, and autoimmune diseases, and are utilized as an important tool for evaluating the efficacy and safety of new drugs. Furthermore, because they mimic the human immune system, they are useful for studying the biology of human hematopoietic stem cells and hematopoietic reconstitution after transplantation, as well as for vaccine development and immunotherapy research. In particular, they play an important role as a preclinical model for screening anticancer treatments and applying gene therapy. As a preclinical model, humanized mice are essential for preclinical testing of gene therapies using retroviral vectors or gene editing tools for various indications, including severe combined immunodeficiency (SCID), X-linked severe combined immunodeficiency (SCID-X), chronic granulomatous disease (X-CGD), hemoglobinopathies, and hereditary bone marrow failure syndromes (IBMFS).

[0003] The creation of a humanized mouse model using CD34-positive hematopoietic stem cells is mainly accomplished by injecting human CD34-positive hematopoietic stem cells into mice with a deficient immune system. During this process, the hematopoietic stem cells migrate to the bone marrow of the mouse, and the mouse's immune system is replaced with human immune cells. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, functional human antibodies, and cellular immune responses, resulting in an immune response similar to that of a human.

[0004] OP9 stromal cells are bone marrow fibroblasts derived from mice. They are a stromal cell line generated from neonatal (C57BL / 6xC3H)F2-op / op mice deficient in macrophage-colony stimulating factor (M-CSF). They are used to increase the survival of hematopoietic progenitor cells derived from human embryonic stem cells and as feeder cells to differentiate the stem cells into megakaryocytes and platelets, and are used in hematopoietic stem cell differentiation research. By co-culturing OP9 cells with CD34-positive hematopoietic stem cells, hematopoietic stem cells can differentiate into various blood cells under specific conditions, and are particularly useful for studying the differentiation of immune cells.

[0005] The co-culture system using OP9 cells is an effective method for studying hematopoietic stem cell differentiation, but the interaction between OP9 cells and CD34-positive hematopoietic stem cells is very complex, and slight differences in culture conditions can have a great impact on the results of cell differentiation, making it difficult to obtain consistent experimental results. In addition, it is difficult to optimize various conditions such as culture medium, cell density, and cytokine addition in the co-culture system using OP9 cells, so the OP9 cell co-culture system has many problems in establishing optimized culture conditions.

[0006] In addition, although the co-culture method using OP9 cells may be suitable for small-scale laboratory use, it may be inefficient for large-scale cell production, and the expansion of the co-culture system is necessary to obtain a large number of differentiated cells. In addition, the study of differentiation of CD34-positive hematopoietic stem cells using OP9 cells is making an important contribution to the study of hematopoietic diseases, immune responses, and regenerative medicine, so the development of a more efficient and reproducible cell differentiation method is necessary.

[0007] The purpose of the present invention is to provide a method for producing CD34-positive hematopoietic stem cells derived from human pluripotent stem cells using a secretory protein of OP9 cells and a humanized mouse transplanted with CD34-positive hematopoietic stem cells produced by the method.

[0008] To achieve the above purpose, the present invention

[0009] (a) a step of culturing human pluripotent stem cells (hPSCs) to form aggregates;

[0010] (b) a step of differentiating the aggregate into mesodermal cells; and

[0011] (c) comprising a step of differentiating the mesodermal cells into CD34 positive hematopoietic stem cells,

[0012] The above steps (a), (b) and (c) provide a method for producing CD34 positive hematopoietic stem cells, wherein the steps are performed in a medium containing an OP9 cell culture medium containing a secretory protein of OP9 cells.

[0013] In addition, the present invention provides a humanized mouse into which CD34 positive hematopoietic stem cells manufactured by the above manufacturing method are transplanted.

[0014] The method for producing CD34-positive hematopoietic stem cells according to the present invention overcomes the limitations of complex OP9 cell co-culture techniques using OP9 cell culture medium, forms aggregates of human pluripotent stem cells, and enables mass production of hematopoietic stem cells. In addition, growth factors are added step-by-step at each differentiation stage to increase culture efficiency, thereby maximizing the production efficiency of CD34-positive cells. Therefore, by transplanting hematopoietic stem cells produced by the method according to the present invention into a humanized mouse, a model having an immune system similar to the human immune system can be obtained.

[0015] Figure 1 shows a comparison of the degree of differentiation of CD34 positive cells after culturing hiPSCs alone, hiPSCs + OP9 cells directly co-cultured, and hiPSCs + OP9 cells separately co-cultured for CD34 positive hematopoietic stem cell differentiation according to one embodiment of the present invention.

[0016] Figure 2 shows the degree of differentiation of CD34 positive hematopoietic stem cells cultured for 10 days using hiPCS aggregates and OP9 cells prepared as a collagen mixed patch according to one embodiment of the present invention.

[0017] Figure 3 shows the results of analyzing the secreted protein of OP9 cells through a 3-day culture medium according to one embodiment of the present invention.

[0018] Figure 4 shows the results of analyzing the secreted protein of OP9 cells through a 6-day culture medium according to one embodiment of the present invention.

[0019] Figure 5 shows the results of comparing hematopoietic stem cell differentiation-related proteins among the secreted proteins of OP9 cells according to one embodiment of the present invention through 3-day culture and 6-day culture.

[0020] Figure 6 is a graph comparing hematopoietic stem cell differentiation-related proteins among secreted proteins of OP9 cells according to one embodiment of the present invention through 3-day culture and 6-day culture.

[0021] Figure 7 is a schematic diagram showing a method for producing CD34 positive hematopoietic stem cells using a secreted protein of OP9 cells as a conditioned medium (CM) according to one embodiment of the present invention.

[0022] Figure 8 shows a comparison of the degree of differentiation of CD34 positive hematopoietic stem cells according to the inclusion of a conditioned medium (CM) and growth factors according to one embodiment of the present invention.

[0023] Hereinafter, the present invention will be described in detail.

[0024] The present invention comprises the steps of (a) culturing human pluripotent stem cells (hPSCs) to form aggregates;

[0025] (b) a step of differentiating the aggregate into mesodermal cells; and

[0026] (c) comprising a step of differentiating the mesodermal cells into CD34 positive hematopoietic stem cells,

[0027] The above steps (a), (b) and (c) provide a method for producing CD34 positive hematopoietic stem cells, wherein the steps are performed in a medium containing an OP9 cell culture medium containing a secretory protein of OP9 cells.

[0028] The above human pluripotent stem cells may be human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), preferably human induced pluripotent stem cells (hiPSCs).

[0029] In the present invention, the OP9 cell culture medium is a "conditioned media (CM)" that can support the growth and survival of cells in vitro, and may refer to a culture medium containing growth factors optimized for differentiation of human pluripotent stem cells of the present invention into CD34 positive hematopoietic stem cells. The OP9 cell culture medium may include a secreted protein contained in a culture medium obtained after 3 or 6 days of co-culturing human pluripotent stem cells and OP9 cells.

[0030] The above secreted protein is a hematopoietic growth factor that stimulates the growth and differentiation of hematopoietic stem cells and may include cytokines and growth factors. The cytokine may include one or more cytokines from the group consisting of leukemia inhibitory factor (LIF), interleukin-3 (IL-3), IL-6, IL-7, IL-11, IL-15, erythropoietin (EPO), and thrombopoietin (TPO), and the growth factor may include epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor (IGF-2), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell It may contain one or more growth factors from the group consisting of growth factors (SCF) and Flt3-L (FMS-like tyrosine kinase 3 Ligand).

[0031] In a specific embodiment of the present invention, the secreted protein may be one or more proteins selected from the group consisting of G-CSF, IL-6, M-CSF, SCF, TPO, VEGF, Flt-3L, IGF-1 and IL-11, preferably IL-6, M-CSF, SCF, VEGF, IGF-1 and IL-3, more preferably G-CSF, IL-6, M-CSF, SCF, TPO, VEGF, Flt-3L, IGF-1 and IL-11.

[0032] The above secreted protein may be included in the culture medium obtained 3 or 6 days after co-culture of human pluripotent stem cells with OP9 cells.

[0033] The OP9 cell culture medium may be present in an amount of 10 to 80 wt%, preferably 25 to 75 wt%, and more preferably 50 wt%, based on the total weight of the medium composition.

[0034] The above co-cultured OP9 cells may be cultured in the form of a collagen mixed patch to suppress OP9 cell migration and increase the number of secreting cells.

[0035] The above collagen may be any one selected from the group consisting of collagen type I, collagen type II, collagen type III, collagen type IV, and collagen type V, and preferably collagen type I.

[0036] The medium for the above steps (a), (b), (c) and co-culture may be a culture medium known in the art that can support the growth and survival of cells in vitro, and examples thereof include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), CCMM (cell culture minimum medium), and IMDM (Iscove's Modified Dulbecco's Medium), but is not limited thereto and the medium and culture conditions may be selected depending on the type of cell, and preferably may be DMEM.

[0037] The medium of the above steps (a), (b), (c) and co-culture may contain 1 to 10 wt% FBS (fetal bovine serum), preferably 10 wt% FBS.

[0038] The above steps (a), (b), (c) and co-culture may use a culture vessel coated with Matrigel, fibronectin, laminin or Pluronic F-127 to prevent cell attachment.

[0039] The aggregates formed in the above step (a) may play a role in promoting mass production of hematopoietic stem cells and differentiation of hematopoietic stem cells.

[0040] The above step (a) may be performed in a medium to which CHIR99021 is additionally added.

[0041] CHIR99021 of the above step (a) may be added to the medium at 1 to 10 μM, preferably 4 to 8 μM, or more preferably 6 μM.

[0042] The above step (a) may be performed for 1 to 4 days, preferably for 2 days.

[0043] The above step (b) may be performed in a medium to which BMP4 is additionally added.

[0044] The BMP4 of the above step (b) may be added at 30 to 70 ng / ml, preferably 40 to 60 ng / ml, or more preferably 50 ng / ml.

[0045] The EPO of the above step (b) may be added at 30 to 70 ng / ml, preferably 40 to 60 ng / ml, or more preferably 50 ng / ml.

[0046] The above step (b) may be performed for 1 to 4 days, preferably 2 days.

[0047] The above step (c) is a differentiation step in which the mesodermal cells differentiate into hematopoietic progenitor cells; and

[0048] The above hematopoietic progenitor cells may include a maturation stage in which they mature into CD34 positive hematopoietic stem cells.

[0049] The differentiation step of the above step (c) may be performed in a medium to which BMP4 and EPO are additionally added.

[0050] The maturation step of the above step (c) may be performed in a medium to which BMP4, EPO and c-Kit are additionally added.

[0051] The concentrations of BMP4 and EPO added in the differentiation and maturation stages of the above step (c) may be the same.

[0052] BMP4 in the differentiation and maturation stages of the above step (c) may be added at 30 to 70 ng / ml, preferably 40 to 60 ng / ml, or more preferably 50 ng / ml.

[0053] EPO in the differentiation and maturation stages of the above step (c) may be added at 30 to 70 ng / ml, preferably 40 to 60 ng / ml, or more preferably 50 ng / ml.

[0054] The C-Kit of the above step (c) may be added at 10 to 30 ng / ml, preferably 15 to 25 ng / ml, or more preferably 20 ng / ml.

[0055] The above step (c) may be performed for 2 to 8 days, preferably 4 days.

[0056] In the present invention, the rate at which the hematopoietic progenitor cells mature into the CD34 positive hematopoietic stem cells may be at least 20% or more, preferably 20 to 30%.

[0057] In addition, the present invention provides a humanized mouse into which CD34 positive hematopoietic stem cells manufactured by the above manufacturing method are transplanted.

[0058] In a specific embodiment of the present invention, it was confirmed that the differentiation efficiency of CD34-positive cells was improved through co-culture of human pluripotent stem cells and OP9 cells (Fig. 1), and the differentiation efficiency of CD34-positive cells was further improved through culture using OP9 cell collagen mixed patches and human pluripotent stem cell aggregates (Fig. 2), and the culture medium with improved differentiation efficiency was analyzed to confirm that the amount of hematopoietic stem cell differentiation-related proteins increased in the 6-day culture medium (Figs. 3 to 6). In addition, it was confirmed that the complex OP9 cell co-culture technique was overcome through the 6-day culture medium of OP9 cells, and the differentiation efficiency of CD34-positive hematopoietic stem cells was increased by adding growth factors at the differentiation stage of hematopoietic stem cells (Figs. 7 to 8).

[0059] Therefore, the efficiency of humanized mouse production can be improved by using CD34 positive hematopoietic stem cells manufactured by the manufacturing method of the present invention.

[0060] Furthermore, since the humanized mouse of the present invention is transplanted with hematopoietic stem cells produced using OP9 cell culture medium that maximizes the production efficiency of CD34-positive cells, the humanized mouse functions as a model with an immune system similar to the human immune system. Furthermore, the model can be developed into a patient-specific immunohumanized mouse and useful for research on disease models, customized cell therapy, and immuno-oncology therapy.

[0061] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0062] The terms used in the examples are for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprising," "included," or "having" specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0063] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0064] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are intended to specifically illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0065] Example 1. CD34-positive cell production strategy

[0066] 1.1 Exploration of OP9 cell coculture techniques

[0067] Stem cells were cultured for 10 days using a co-culture technique using OP9 cells, and the production efficiency of CD34-positive cells was analyzed.

[0068] For analysis, OP9 cells (CRL2749-ATCC) were directly co-cultured and isolated co-cultured with human induced pluripotent stem cells (hiPSCs, produced by Chung-Ang University).

[0069] Direct co-culture was performed by mixing OP9 cells and hiPSCs in a medium, and isolated co-culture was performed by isolating OP9 cells and hiPSCs using a Co-culture insert (Thermo Fisher Scientific, pore size: 0.4 ㎛). The medium was Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (Hyclone), and the cells were cultured for 10 days under standard co-culture conditions (37°C, 5% CO2, 95% humidity), and then analyzed by FACS.

[0070] Comparing hiPSCs alone and hiPSCs + OP9 cell co-culture (direct co-culture and separate co-culture), it was observed that direct co-culture (10.78%) and separate co-culture (8.25%) had higher CD34-positive cell differentiation rates than single culture (2.44%), indicating that the efficiency of CD34-positive cell production increased under OP9 cell co-culture conditions (Fig. 1).

[0071] 1.2 Optimization of OP9 cell coculture technique

[0072] To explore the optimization of the OP9 cell co-culture technique, OP9 cells were prepared in the form of patches mixed with collagen, and hiPSC aggregates were used.

[0073] Specifically, collagen patches (SpongeCol ® #5135, ADVANCED Biomatrix) to produce a patch by adsorbing OP9 cells, and hiPSCs were cultured in a labsparrow cell culture dish (labsparrow) that can form aggregates. TM , (주)랩투랩) was used to manufacture and use aggregates.

[0074]

[0075] *The fabricated collagen mixed patch and hiPSC aggregates were separated and co-cultured in DMEM medium containing 10% FBS. The medium was replaced every two days, and the culture dish was coated with Pluronic F-127 (Sigma) to prevent cell attachment.

[0076] As a result of analyzing the ratio of differentiated CD34 positive cells after 10 days of culture, a CD 34 positive cell differentiation ratio of 12.62% was observed, confirming that the differentiation efficiency was increased compared to the co-culture of Example 1.1 (Fig. 2).

[0077] 1.3 Analysis of 0P9 cell secreted proteins

[0078] In the above Example 1.2, the culture medium components were analyzed on each differentiation day (day 3 and day 6) to identify proteins related to hematopoietic stem cell differentiation.

[0079] Analysis was performed using RayBio, a cytokine analysis device. ® The mouse cytokine array C2000 kit (RayBiotech, Noncross, GA, USA) was used, and the analysis samples were cultured OP9 cells in DMEM medium for 3 or 6 days, and the results for CYT 3, 4, and 5 are shown in Tables 1 to 3 (3-day culture) and Tables 4 to 6 (6-day culture), respectively.

[0080] 1) Protein analysis of 3-day culture fluid

[0081] 번호분비단백질단백질 발현양1CXCL 1612,545.752IL-1alpha373.253IL-6210,774.754KC147,453.755LIX172,815.256MCP-1360,912.257M-CSF2,871.758MIP-1 gamma160,187.259MIP-272,477.2510PF4828.7511P-Selectin679.2512RANTES129,366.2513SCF1,434.7514SDF-1-alpha143,727.2515TCA-31,342.7516TIMP-153,990.2517sTNF-R1 (Soluble TNF Receptors 1)194,427.7518sTNF-R2 (Soluble TNF Receptors 2)51,668.2519VCAM-150,377.2520VEGF11,754.75

[0082] 번호분비단백질단백질 발현양1ICAM-111,643.252IGFBP-21,069.753IGF-1515.254Lungkine707.755MMP-240,343.256MMP-330,588.257Osteopontin80,880.758Osteoporotegerin45,076.759Resistin3,873.7510Thymus CK-1757.75

[0083] NumberSecreted proteinProtein expression amount16Ckine129.252ALK-125.753Amphiregulin289.254CD362,058.255CD40 Ligand2,065.756CTLA-4734.257Decorin496.258DKK-1628.259Epiregulin8,581.7510Galectin-1135,070.7511Growth arrest specific 1198,901.7512Growth arrest specific 6236,204.2513GITR Ligand6,786.2514Granzyme B3,059.7515HGF38,463.2516IL-1 R4 / ST2L7,381.7517IL-11274.2518IL-17B313.7519IL-17E3,872.7520IL-1ra / IL-1F337,735.7521IL-21396.7522IL-288,492.7523IL-6 R1,595.7524JAM-A2,699.2525MFG-E817,193.7526Neprilysin18,874.2527Pentraxin 341,467.7528TREM-11,054.2529TWEAK R96,853.25

[0084] As a result of analysis of the 3-day culture fluid proteins, proteins related to hematopoietic stem cell differentiation were identified, including No. 3 (IL-6), No. 7 (M-CSF), No. 13 (SCF), and No. 20 (VEGF) in Table 1, No. 3 (IGF-1) in Table 2, and No. 17 (IL-11) in Table 3 (Fig. 3 A (Table 1), B (Table 2), and C (Table 3)).

[0085] 2) Protein analysis of 6-day culture fluid

[0086] 번호분비단백질단백질 발현양1axl658.272CTACK596.513CXCL1624,895.284Fractalkine867.845G-CSF18,979.416IGF-BP-3860.617IL41,202.888IL6249,381.139IL12-p40 / p703,335.3610IL12-p701,148.3611KC176,947.3312LIX220,768.8013L-Selectin2,309.8514Lymphotactin1,575.3815MCP-1330,451.9016MCP-51,820.4217M-CSF11,995.9818MIP-1-alpha1,607.5719MIP-1-gamma192,295.0920MIP-283,301.1421PF41,864.4422P-Selectin1,409.8223RANTES153,785.7524SCF6,255.5225SDF-1-alpha114,252.8826TCA-33,300.5427TIMP-152,455.8228sTNF R1 (Soluble TNF Receptors 1)214,500.1429sTNF R2 (Soluble TNF Receptors 1)73,391.6430TPO1,089.2331VCAM-155,766.8732VEGF24,835.50

[0087] 번호분비단백질단백질 발현양1Flt-3 Ligand281.632ICAM-122,005.563IGFBP-2697.214IGF-I221.505Lungkine204.196MMP-240,551.537MMP-3134,084.628Osteopontin80,082.619Osteoporotegerin42,926.2310Resistin95,935.9311Thymus CK-1671.24

[0088] NumberSecreted proteinProtein expression amount14-1BB3635.9426Ckine1,862.873ALK-1778.874Amphiregulin1,432.205CD3610,029.246CD40 Ligand1,557.957CTLA-4615.648Decorin1,383.969DKK-12,647.5510Endoglin1,000.2311Epiregulin15,169.3012Galectin-1141,507.8213Growth arrest specific 1223,785.2914Growth arrest specific 6271,800.7515GITR Ligand7,405.1716Granzyme B1,191.4517HGF70,803.7618IL-1 R4 / ST2L20,635.3719IL-112,085.5220IL-17B2,008.8621IL-17E10,431.4922IL-17F1,124.2723IL-1ra / IL-1F321,212.0424IL-2 R alpha855.9625IL-21217.7026IL-287,015.8427IL-6 R276.7128JAM-A1,478.7129MFG-E864,632.6830Neprilysin54,203.5631Pentraxin 372,658.6632Prolactin1,501.9633TACI1,034.2634TREM-14,388.3235TWEAK R98,361.51

[0089] As a result of analysis of the 6-day culture fluid proteins, proteins related to hematopoietic stem cell differentiation were identified, including No. 5 (G-CSF), No. 8 (IL-6), No. 17 (M-CSF), No. 24 (SCF), No. 30 (TPO), and No. 32 (VEGF) in Table 4, No. 1 (Flt-3L), No. 4 (IGF-1) in Table 5, and No. 19 (IL-11) in Table 6 (Fig. 4 A (Table 4), B (Table 5), and C (Table 6)).

[0090] 3) Comparison of OP9 cell secreted proteins

[0091] As a result of comparative analysis of the culture medium, it was confirmed that three types of proteins (G-CSF, TPO, and Flt-3L) were additionally secreted in the 6-day culture medium (Fig. 5), and it was confirmed that the amount of protein was greater in the 6-day culture medium than in the 3-day culture medium (Fig. 6).

[0092] Consequently, the experimental results of Example 1 above suggest that using a 6-day culture of OP9 cells as a conditioned medium (CM) to differentiate hematopoietic stem cells is more effective in improving differentiation efficiency.

[0093] Example 2. Differentiation of CD34-positive hematopoietic stem cells using OP9 cell secreted proteins.

[0094] CD34-positive hematopoietic stem cell culture was performed using a 6-day culture medium containing OP9 cell secretory proteins, which are efficient in producing CD34-positive cells, as a conditioned medium (CM) according to Example 1 above. The culture was performed according to the schematic diagram in Fig. 7, and the medium was replaced every two days at each step.

[0095] hiPSCs (produced by Chung-Ang University) as in Example 1 were cultured in iPS-BREW XF medium (Stem MACS TM , Mitenyi Biotec) and maintained in an undifferentiated state for 2 days.

[0096] To form aggregates, 50% of the conditioned medium (CM) in which OP9 cells were cultured for 6 days in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (hyclone) was added, and the maintained hiPSCs were differentiated for 2 days by treating them with 6 μM of the small molecule compound CHIR99021 (Tocris). The conditioned medium (CM) contains all six hematopoietic stem cell differentiation-related proteins (G-CSF, IL-6, M-CSF, SCF, TPO, VEGF, Flt-3L, IGF-1) analyzed in Example 1.3, and is therefore optimized for differentiation into CD34-positive cells.

[0097] After confirming that aggregates were formed, the aggregates were cultured for 2 days in DMEM medium containing 50% conditioned medium (CM) and 10% fetal bovine serum, treated with 50 ng / ml BMP4 (R&D systems), and differentiated into mesoderm.

[0098] Afterwards, the medium was treated with 50 ng / ml BMP4 and 50 ng / ml EPO (R&D Systems) to confirm the morphology of hematopoietic progenitor cells after 2 days, and then 50 ng / ml BMP4, 50 ng / ml EPO, and 20 ng / ml c-Kit / CD117 (R&D systems) were treated in DMEM medium to induce differentiation into CD34 positive cells and cultured for 2 days.

[0099] As a result of analyzing the cultured cells by separating them using FACS, the ratio of CD34-positive hematopoietic stem cells was confirmed to be 20.84% ​​in the medium treated with conditioned medium (CM) and growth factors at each stage, which was a higher ratio of CD34-positive hematopoietic stem cell differentiation than in the medium containing only conditioned medium (CM) (7.05%) or the medium treated with only growth factors excluding conditioned medium (CM) (5.92%) (Fig. 8).

[0100] Therefore, from the results of the above example, it was confirmed that when a conditioned medium (CM) in which OP9 cells were cultured for 6 days was used, the differentiation efficiency of CD34-positive cells increased with the addition of growth factors at each differentiation stage, and it was confirmed that the conditioned medium (CM) could be used as a differentiation induction method that facilitates differentiation and isolation of CD34-positive hematopoietic stem cells rather than the OP9 cell co-culture method, thereby confirming the possibility of using it in a humanized mouse transplantation model.

Claims

1. (a) A step of forming an aggregate by culturing human pluripotent stem cells (hPSCs); (b) a step of differentiating the aggregate into mesodermal cells; and (c) comprising a step of differentiating the mesodermal cells into CD34 positive hematopoietic stem cells, A method for producing CD34 positive hematopoietic stem cells, wherein the steps (a), (b) and (c) above are performed in a medium containing OP9 culture medium containing secretory proteins of OP9 cells.

2. In paragraph 1, A method for producing CD34 positive hematopoietic stem cells, wherein the above secreted protein is at least one protein selected from the group consisting of G-CSF, IL-6, M-CSF, SCF, TPO, VEGF, Flt-3L, IGF-1, and IL-11.

3. In paragraph 1, A method for producing CD34 positive hematopoietic stem cells, wherein the above step (a) is performed in a medium to which CHIR99021 is additionally added.

4. In paragraph 1, A method for producing CD34 positive hematopoietic stem cells, wherein the above step (b) is performed in a medium to which BMP4 is additionally added.

5. In paragraph 1, The above step (c) is a differentiation step in which the mesodermal cells differentiate into hematopoietic progenitor cells; and A method for producing CD34 positive hematopoietic stem cells, comprising a maturation step in which the above hematopoietic stem cells mature into CD34 positive hematopoietic stem cells.

6. In paragraph 5, A method for producing CD34 positive hematopoietic stem cells, wherein the above differentiation step is performed in a medium to which BMP4 and EPO are additionally added.

7. In paragraph 5, A method for producing CD34 positive hematopoietic stem cells, wherein the above maturation step is performed in a medium to which BMP4, EPO and c-Kit are additionally added.

8. In paragraph 5, A method for producing CD34-positive hematopoietic stem cells, wherein the rate of maturation of the above hematopoietic stem cells into CD34-positive hematopoietic stem cells is at least 20%.

9. A humanized mouse transplanted with CD34 positive hematopoietic stem cells manufactured by the manufacturing method of Article 1.

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