KR20220068305A - Animal model using hematopoietic stem cell derived from induced pluripotent stem cell and use thereof - Google Patents

Abstract

The present invention relates to an animal model using hematopoietic stem cells derived from dedifferentiated stem cells. According to one aspect, provided is a method for analyzing therapeutic efficacy of an anti-cancer drug. The method comprises the steps of: isolating cells from a biological sample; preparing dedifferentiated stem cells (induced pluripotent stem cells: iPSC) from the isolated cells; inducing hematopoietic stem cells (HSC) from the prepared dedifferentiated stem cells; and administering the induced hematopoietic stem cells to an animal so as to prepare an animal model. Accordingly, the animal model prepared thereby can be usefully applied to analyze therapeutic efficacy of a cancer immunotherapy agent.

Description

Animal model using hematopoietic stem cells derived from retrodifferentiated stem cells and uses thereof

It relates to an animal model using hematopoietic stem cells derived from retrodifferentiated stem cells and uses thereof.

In the case of chemotherapy, a number of studies have been conducted to select the most effective anticancer drug because it shows various treatment responses and results according to individual patients even with the same drug. The model using the in vitro culture method has certain limitations, such as excluding interactions with surrounding cells and the surrounding microenvironment is different from that of the human body. is becoming Among them, rodents, especially mice, have been most frequently used as animals for disease model production. In particular, the xenograft model is widely used in the preclinical stage to predict the anticancer effect of an anticancer drug candidate, and is also used to predict the clinical outcome under an appropriate environment.

Hematopoietic stem cells derived from cord blood are often used for transplantation because of their advantages over bone marrow. However, in many transplant cases, the total number of nucleated cells used for transplantation is pointed out as the single most important factor for the success rate of umbilical cord blood stem cell transplantation. The transplant results were not good due to complications due to delay, etc.

Therefore, the possibility of mixed transplantation from multiple allogeneic donors has been raised to increase the total number of cells obtainable from the placenta and to increase the amount of engraftment. However, the clinically tried results of this mixed transplant did not secure a control for the content of transplanted hematopoietic stem cells in each of the two mixed cord blood, but also presented different results in a limited number of clinical cases. are doing

Under this technical background, various studies are being conducted to secure a sufficient number of single-derived hematopoietic stem cells, and to evaluate the effectiveness of drugs such as anticancer drugs by manufacturing an animal model using the same (Korean Patent No. 10-0666607), It is still ambiguous.

One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells (induced Pluripotent Stem Cell: iPSC) from the isolated cells; Inducing hematopoietic stem cells (HSC) from the prepared dedifferentiated stem cells; And to provide a method for producing an animal model, comprising the step of administering the induced hematopoietic stem cells to the animal.

Another aspect is to provide an animal model for analyzing the therapeutic efficacy of an anticancer agent prepared by the above manufacturing method.

Another aspect comprises the steps of administering an anticancer agent to the animal model; And to provide a method for analyzing the therapeutic efficacy of an anticancer agent, comprising the step of determining the therapeutic efficacy of the anticancer agent by analyzing the growth or metastasis of cancer cells in the animal model.

Another aspect includes administering an anticancer drug candidate to the animal model; And analyzing the growth or metastasis of cancer cells in the animal model to provide a screening method for an anticancer agent comprising the step of determining the therapeutic efficacy of the anticancer agent.

One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells from the isolated cells; Inducing hematopoietic stem cells from the prepared dedifferentiated stem cells; And it provides a method for producing an animal model, comprising the step of administering the induced hematopoietic stem cells to the animal.

The "biological sample" refers to a sample derived from an organism. The organism may be a mammal including a human. The biological sample may be derived from a tissue or body fluid of the body. The tissue may be any tissue in the body capable of producing a tumor. The body fluids include blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid , ascites, cystic tumor body fluid, amniotic fluid, or a combination thereof. The blood may be derived from peripheral blood. The blood may include blood cells. The blood cells may include one or more of red blood cells, white blood cells, and platelets. The white blood cells may include eosinophils, basophils, neutrophils, lymphocytes, monocytes, macrophages, and the like. The cells isolated from the biological cells may be mononuclear cells.

The biological sample may be derived from a cancer patient. Accordingly, the animal model can be usefully used to select a customized drug that can exhibit an effective effect on cancer treatment of a patient by mimicking the immune system of a specific patient.

The “induced pluripotent stem cell (iPSC)” is also referred to as an induced pluripotent stem cell, and refers to a cell having pluripotency obtained by de-differentiation from a differentiated cell (eg, a somatic cell). do. The dedifferentiated stem cells can differentiate into various organ cells. The dedifferentiated stem cells can be obtained by reprogramming the cells differentiated by the dedifferentiation 　inducing factors.

The "hematopoietic stem cell (HSC)" is used interchangeably with 　 hematopoietic stem cell, and refers to a cell that performs hematopoietic action through self-proliferation and differentiation. The hematopoietic stem cells mature into final cells through progenitor cells, which are intermediate processes of various blood cells, and various types of blood such as red blood cells, platelets, neutrophils, eosinophils, basophils, monocytes, T cells, B cells, natural killer cells, or dendritic cells. Since it differentiates into internal cells, the development of red blood cells or lymphocytes can be promoted by the differentiation of the hematopoietic stem cells. The 　 hematopoietic stem cells have the ability to differentiate into various blood cells by a specific differentiation-inducing stimulus as well as self-renewal capacity in an undifferentiated state. The hematopoietic stem cells may be derived from any animal such as humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, and the like.

In one embodiment, to differentiate or induce the dedifferentiated stem cells into hematopoietic stem cells may include the following steps:

(a) culturing the dedifferentiated stem cells in a medium to which bone morphogenetic protein 4 (BMP4) is added;

(b) culturing by replacing the cells cultured in step (a) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added;

(c) culturing the cells cultured in step (b) by replacing them with a medium to which SB431542, vascular endothelial growth factor, and basic fibroblast growth factor are added;

(d) culturing by replacing the cells cultured in step (c) with a medium to which stem cell factor (SCF), thrombopoietin (TPO), and interleukin-3 (IL-3) are added;

(e) obtaining a culture supernatant of the cells cultured in step (d);

(f) separating the cells contained in the obtained supernatant; and

(g) the isolated cells Flt3L (FMS-like tyrosine kinase 3 ligand), stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), and interleukin-6 (IL-6) ) culturing in the added medium.

In one embodiment, the hematopoietic stem cells may express one or more markers selected from the group consisting of CD34, CD43 and CD45. The hematopoietic stem cells may express two or more selected from the group consisting of CD34, CD43 and CD45. The hematopoietic stem cells may express CD34, CD43 and CD45. For example, the hematopoietic stem cells may express CD34 and CD45. For example, the hematopoietic stem cells may express CD43 and CD45.

The "administration" may be used interchangeably with "injection", "injection" or "infection".

The "administering step" includes administering to an animal in any manner. For example, the induced hematopoietic stem cells may be administered by intravenous, intramuscular, subcutaneous, rectal, intraperitoneal, intrauterine intrathecal or intracerebrovascular injection, or may be administered orally. Preferably, the induced hematopoietic stem cells may be injected intravenously into animals.

The "animal" includes, without limitation, any animal that can be used as an animal model, preferably a mammal. The mammal may be a mammal other than a human. Specifically, the animal may be one selected from the group consisting of a mouse, a rat, a guinea pig, a monkey, a dog, a cat, a rabbit, a cow, a sheep, a pig, and a goat. Among the animals, mice have good reproductive ability, developmental ability, and environmental adaptability, and have a large number of progeny, so that a large number of individuals belonging to the experimental group can be secured, thereby obtaining reliable results and easy operation. In addition, the humanized mouse is known to be 97% similar to human genes, and is useful as a model for replacing humans.

The animal may be an immunodeficient animal. The term "immune deficient animal" refers to an animal with reduced or deficient immune response due to various causes such as reduction, deficiency or dysfunction of T cells, B cells, macrophages, antibodies, complement, etc. involved in the immune response. . The immunodeficient animal may be an animal deficient in at least one selected from the group consisting of T cells, B cells, and natural killer (NK) cells. The immunodeficient animal may be a severe combined immunodeficient syndrome (SCID) mouse, a SCID-beige mouse, a NOD/Shi-scid/IL-2Rγnull (NOG) mouse, and a NOD scid gamma (NSG) mouse. . The severe combined immunodeficiency mouse is a mutant mouse having severe combined immunodeficiency syndrome, in which both cellular and humoral immunity are deficient due to birth defects in both T cells and B cell lines due to a developmental disorder of lymphocyte stem cells. can mean The SCID-beige mouse may have autosomal recessive mutations in both SCID and Beige. In the case of the SCID mutation, T cells and B cells are absent as described above, and in the case of the Beige mutation, a deficiency of NK cells may appear. The SCID-beige mouse exhibits the above characteristics at the same time, and may be deficient in T cells, B cells and NK cells. The Nog mice may be double homozygous for a severe combined immunodeficiency mutation and an interleukin-2R (IL-2Rγ) allele mutation, developed as a receptor for xenografts. The Nog mouse may refer to an immunodeficient mouse deficient in NK cells in addition to T cells and B cells. The NSG mouse may be a cross between a severe combined immunodeficiency mouse and a non-obese diabetic (NOD) mouse. The NSG mice do not have mature T cells and B cells at all, and show very low NK cell activity, so the transplantation efficiency of human cells may be high. The immunodeficient animal may be an NSG mouse. The immunodeficient animal may be an NSG-A mouse.

The "administration step" may use any animal of any age suitable for the purpose. For example, the animal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks of age. can In addition, the animal may be an animal of 1 to 20, 1 to 10, 1 2 to 10, 2 to 8, 2 to 6, 3 to 6, 3 to 5, 3 to 4, 4 to 5 weeks of age.

In one embodiment, the induced hematopoietic stem cells may be administered in any amount necessary to prepare the animal model of the present specification. For example, the hematopoietic stem cells are 1x10 ⁵ to 1x10 ⁷ per animal, 2x10 ⁵ to 1x10 ⁷ , 4x10 ⁵ to 1x10 ⁷ , 6x10 ⁵ to 8x10 ⁷ , 1x10 ⁵ to 8x10 ⁶ , 1x10 ⁵ to 6x10 ⁶ , 1x10 ⁵ to 4x10 ⁶ , 1x10 ⁵ to 2x10 ⁶ , 2x10 ⁵ to 2x10 ⁶ , 4x10 ⁵ to 2x10 ⁶ , 6x10 ⁵ to 2x10 ⁶ , 7x10 ⁵ to 2x10 ⁶ , 1x10 ⁶ to 2x10 ⁶ The number of cells may be administered. Preferably, the hematopoietic stem cells may be administered to the animal in an amount of 1x10 ⁶ cells per animal.

The animal may be irradiated with radiation. The radiation may be irradiated at any stage prior to administration of the induced hematopoietic stem cells to the animal. For example, the radiation may be used before isolating cells from a biological sample, before preparing dedifferentiated stem cells from the isolated cells, before inducing hematopoietic stem cells from the prepared dedifferentiated stem cells, or injecting the induced hematopoietic stem cells into an animal. The animal may be irradiated prior to administration.

In one embodiment, irradiation may be a method for preventing host-vs-graft (HVG) rejections. The irradiation may be to suppress the host immune system in order for the human immune system to survive successfully in the animal. The radiation can be used to clear the bone marrow niche of host progenitor cells. The radiation can remove the bone marrow fissure to allow space for donor stem cell engraftment. The irradiation may be by whole body or partial gamma irradiation.

The radiation dose may be any suitable amount for preparing the animal model. For example, the radiation dose is 0.5 to 10 Gy, 1 to 10 Gy, 1.5 to 10 Gy, 0.5 to 7 Gy, 0.5 to 5 Gy, 0.5 to 3 Gy, 0.5 to 2 Gy, 0.5 to 1.5 Gy, 1 to 5 Gy, 1 to 3 Gy, 1 to 2 Gy, 1 to 1.5 Gy or 1.5 to 2 Gy.

Another aspect provides an animal model for analyzing the therapeutic efficacy of an anticancer agent prepared by the manufacturing method.

The term animal model and the like are the same as described above.

The “anti-cancer agent” may be any substance known to treat cancer. The cancer is also called a malignant tumor, and breast cancer, chronic myelogenous leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, lung cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer ; , prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, lymphocyte lymphoma, kidney cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem nerve It may be a glioma or a pituitary adenoma.

The anticancer agent may be a chemical anticancer agent, a targeted anticancer agent, or an immune anticancer agent, but is not limited thereto. The anticancer agent may be selected according to the type of cancer, the degree of progression, the condition of the patient, and the like, and two or more anticancer agents may be used simultaneously.

The "immune anticancer drug" refers to a class of drugs that treat cancer by preventing cancer cells from evading the body's immune system or by allowing immune cells to better recognize cancer cells. The immuno-cancer agent is a PD-1 inhibitor or a PD-L1 inhibitor such as nivolumab, pembrolizumab, RN888 (anti-PD-1 antibody), atezolizumab (PD-L1-specific mAb from Roche), Durvalumab (a PD-L1-specific mAb from Astra Zeneca), and Avelumab (a PD-L1-specific mAb from Merck). Since the animal model mimics human blood cells, in particular, it can be usefully utilized to analyze the therapeutic efficacy of immune anticancer drugs.

The anticancer agent may be administered orally, parenterally, intraarterially, intradermally, transdermally, intramuscularly, intraperitoneally, intravenously, subcutaneously or intranasally to an animal model in order to analyze the therapeutic efficacy, but is not limited thereto.

The "therapeutic efficacy" may be analyzed by methods commonly used in the art. For example, the therapeutic efficacy may be analyzed with the naked eye, microscopic or endoscopy, ultrasound, surgical operation, biopsy, or tissue staining. In addition, the therapeutic efficacy may be analyzed by measuring the expression level of a related gene or microvascular density according to the type of cancer, or it may be measured by observing the growth or metastasis of cancer cells or using a tool such as a caliper. For example, when further growth or metastasis of cancer cells is inhibited, it may be determined that the anticancer agent to be analyzed has efficacy as an anticancer agent. The therapeutic efficacy may be appropriately determined by a person skilled in the art according to specific purposes, and may be compared with a control group not administered with an anticancer agent for appropriate judgment of the therapeutic efficacy.

The animal model shows that human CD45 is present in 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of leukocytes in the blood; It may be expressed in 75%, or 80% or more. The CD45 has been known as a leukocyte common antigen, Ly-5, T200, B220, and the like. The CD45 may be expressed as a cell surface antigen in all blood nucleated cells except for red blood cells and platelets, and specifically, may be widely present in immune-related cells such as T lymphocytes, B lymphocytes, macrophages, granulocytes, and thymocytes.

Another aspect is the step of administering an anticancer agent to the animal model; and analyzing the growth or metastasis of cancer cells in the animal model to determine the therapeutic efficacy of the anticancer agent.

The term animal model, anticancer agent, cancer, therapeutic efficacy, etc. are the same as described above.

Another aspect includes administering an anticancer drug candidate to the animal model; and analyzing the growth or metastasis of cancer cells in the animal model to determine the therapeutic efficacy of the anticancer agent.

The term animal model, anticancer agent, cancer, therapeutic efficacy, etc. are the same as described above.

The "anticancer drug candidate" refers to an unknown substance used in screening to examine whether or not it has inhibitory activity against the growth or metastasis of tumor cells. The candidate substances include, but are not limited to, chemicals, peptides, proteins, antibodies, and natural extracts. Candidates analyzed by the screening methods of the present invention are single compounds or mixtures of compounds (eg, natural extracts or cell or tissue cultures). Candidates can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Libraries of synthetic compounds are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) and MycoSearch (USA). Candidate substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution It can be obtained by the required synthetic library method, the 1-bead 1-compound library method, and the synthetic library method using affinity chromatography selection. Methods for synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 may be used, but is not limited thereto.

Since the animal model according to an aspect includes human-derived hematopoietic stem cells, it can mimic the human immune system. Therefore, the animal model can be usefully used to analyze the therapeutic efficacy of an anticancer agent, specifically, an immune anticancer agent.

In addition, while the prior art umbilical cord blood-derived hematopoietic stem cells have problems such as engraftment failure and engraftment delay due to insufficient total cell number during transplantation, hematopoietic stem cells according to an aspect are derived from dedifferentiated stem cells, so the total number of cells is limited. There is an advantage that there is no such thing, whereby there is no need for mixed transplantation, so the possibility of a graft-versus-host reaction can be remarkably low.

When the hematopoietic stem cells according to an aspect are prepared from dedifferentiated stem cells derived from a patient, the animal model has the patient's immune system, so the human leukocyte antigen (HLA) and the like are the same as the patient, and a customized anticancer agent effective for the patient may be useful in determining

According to one embodiment, the animal model can be usefully used clinically to evaluate the effectiveness of an anticancer agent because the production success rate of the animal model is higher than that of the prior art.

1 is a photograph of observing the appearance of HSC cells over time after differentiation of dedifferentiated stem cell-derived hematopoietic stem cells according to an embodiment.
2 is a graph analyzing markers of HSC cells on day 19 after differentiation of dedifferentiated stem cell-derived hematopoietic stem cells according to an embodiment.
3 is a graph confirming the marker human CD45 in humanized mouse blood prepared with dedifferentiated stem cell-derived hematopoietic stem cells according to an embodiment.
4 is a table confirming human CD45, a marker, in humanized mouse blood prepared with dedifferentiated stem cell-derived hematopoietic stem cells according to an embodiment.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of retrodifferentiated stem cell-derived hematopoietic stem cells

1-1. Production of dedifferentiated stem cells from normal human peripheral blood mononuclear cells

In order to prepare dedifferentiated stem cells, mononuclear cells were isolated from the peripheral blood of two normal donors who did not have a disease with the approval of the institutional IRB. To isolate mononuclear cells, blood was centrifuged at 2,000 rpm for 30 minutes using Ficoll Paque-PLUS (GE Healthcare) to separate mononuclear cells, and the separated mononuclear cells were StemSpan-ACF (Stem Cell) with StemSpan cc110 added. Technologies) for 4 days. Then, monocytes were re-inoculated into a 24-well plate coated with vitronectin, and dedifferentiated stem cells were prepared using the CytoTune®2.0 Reprogramming kit (Invitrogen). 12 days after transduction and after retrodifferentiated stem cell colonies are formed, colonies are individually inoculated into vitronectin-coated plates by manual picking, and retrodifferentiated stem cells are inoculated with mTeSR1 medium (Stem Cell Technologies). Incubated and expanded for characterization. Through this, two types of dedifferentiated stem cells from the KW group (KW, c1, p-27) and the BM group (BM, No-2, c1, P-14) were obtained from two normal donors, respectively.

1-2. Preparation of hematopoietic stem cells from retrodifferentiated stem cells

To prepare dedifferentiated stem cell-derived hematopoietic stem cells (iPSC-HSC), mTeSR1 medium was added to a 24-well plate coated with vitronectin, and the KW group of dedifferentiated stem cells prepared in the same manner as in Example 1-1 and Each BM group was inoculated at a concentration of 1 X 10 ⁴ cells and then cultured for 24 hours. Then, 50 ng/mL of bone morphogenetic protein 4 (Bone morphogenetic protein 4: BMP4) was replaced with STEMdiff™ APEL™ 2 medium (Stem cell Technologies, Cat# 05270) and cultured for 2 days. Thereafter, 50 ng/mL of vascular endothelial growth factor (VEGF) and 50 ng/mL of basic fibroblast growth factor (bFGF) were added to the STEMdiff™ APEL™ 2 medium. Incubated for an additional day. The medium was replaced with STEMdiff™ APEL™ 2 medium supplemented with 5 μM of SB431542, 50 ng/mL of VEGF, and 50 ng/mL of bFGF, and further cultured for 2 days, followed by 50 ng/mL of stem cell factor (Stem cell factor: SCF), 50 ng/mL Thrombopoietin (TPO), and 50 ng/mL Interleukin-3 (Interleukin-3: IL-3) supplemented with STEMdiff™ APEL™ 2 medium for 2 days incubated during The culture was carried out for 13 days with replacement of fresh STEMdiff™ APEL™ 2 medium supplemented with 50 ng/mL of SCF, 50 ng/mL of TPO, and 50 ng/mL of IL-3 every two days. The supernatant was collected and centrifuged at 300 g for 10 minutes to obtain cells released into the supernatant, and then, based on StemSpan™ SFEM II medium (Stem Cell Technologies, Cat# 09605), StemSpan™CC110 (Stem cell Technologies, Cat#) 02697), or 10 ng/mL of Flt3L (FMS-like tyrosine kinase 3 ligand), 10 ng/mL of SCF, 10 ng/mL of TPO, 10 ng/mL of IL-3, and 10 ng/mL of mL of a combination of interleukin-6 (Interleukin-6: IL-6) was added, and each medium was re-inoculated and further cultured for 6 days. The medium was replaced with fresh medium every two days during incubation. The overall experimental method is shown in FIG. 1 . The generation of dedifferentiated stem cell-derived hematopoietic stem cells was observed daily through an optical microscope.

As a result, both the KW group and the BM group grew in the form of retrodifferentiated stem cell colonies until day 5, and then started to change form after day 7. In addition, although undifferentiated endothelial spread was seen until day 14, it was confirmed that all of them were differentiated into single-cell type hematopoietic stem cells after day 17. In particular, when cultured in a medium supplemented with Flt3L, SCF, TPO, IL-3, and IL-6, it was confirmed that the differentiation efficiency into higher hematopoietic stem cells was further improved.

Example 2. IPSC derived Hematopoietic stem cells (iPSC-HSC) marker expression analysis

It was attempted to determine whether HSCs were differentiated. Specifically, with respect to iPSC-HSC, the expression of CD34, CD43, and CD45, which are known markers of HSC, was investigated. In order to confirm the cells cultured with iPSC-HSC for about 13 and 19 days by flow cytometry, the monoclonal antibody anti-CD34-PE (IgG1,κ, eBioscience, San Diego, CA, USA), anti-CD43-APC (IgG1, κ, eBioscience), and anti-CD45-FITC (IgG1, κ, eBioscience) antibodies. After washing the cells with a buffer, the fluorescence was measured. In addition, the appropriate isotype antibody was stained and compared. Cell markers were analyzed with a flow cytometer (FACS Calibur; Becton Dickinson, San Diego, CA).

As a result, as shown in FIGS. 1 and 2 , it was confirmed that on the 19th day after differentiation, 87.4% of CD43(+) and CD45(+) cells and 76.3% of CD34(+) and CD45(+) cells. Dedifferentiated stem cell-derived hematopoietic stem cells for which the marker was identified were used to induce humanized mice.

Example 3. Method for manufacturing a humanized mouse using dedifferentiated stem cell-derived hematopoietic stem cells

It was attempted to prepare a humanized mouse using the prepared dedifferentiated stem cell-derived hematopoietic stem cells. Specifically, to induce humanized mice, radiation (1.5 Gy) was applied to NSG-A, 4-week-old mice. Differentiated iPSC-iHSCs were intravenously injected into mice at a cell number of 1x10 ⁶ per animal. 16 weeks after injection, orbital blood was collected from mice, and CD45+ (%), a human immune cell marker, was analyzed as FACs in the collected mouse blood.

For FACs analysis, 5 mL of RBC lysis buffer (#420302, Biolegend) was added to 200 uL of mouse blood obtained through orbital blood sampling from the prepared humanized mice, and reacted on ice for 20 minutes. After the reaction, PBS was filled in the tube, centrifuged at 350 g for 5 minutes, and washed twice. The pellet was then resuspended in FACs buffer. To check whether human immune cells are expressed, Anti-Human CD45-eFluor450 (eBioscience) antibody was added and reacted at 4 °C for 20 minutes. Then, PBS was filled in the FACs tube, centrifuged at 350 g for 3 minutes, and washed twice. After the cells were resuspended in 200 uL of fixation buffer, cell markers were checked by flow cytometry (FACS Calibur; Becton Dickinson, San Diego, CA).

As a result, as shown in FIGS. 3 and 4 , it was confirmed that all of the prepared humanized mice had CD45(+) 25% or more, which is the standard for humanized mice. That is, it was confirmed that all the prepared mice were induced into humanized mice by the dedifferentiated stem cell-derived hematopoietic stem cells.

Example 4. Confirmation of anticancer drug efficacy evaluation effect

It was attempted to determine whether the prepared humanized mouse can evaluate the effectiveness of anticancer drugs.

Specifically, 100 μL of 5x10 ⁶ cells/mL breast cancer cell line HCC-1954 was taken and mixed with 100 μL of Matrigel, and a total of 200 μL was injected subcutaneously using a 1 mL syringe on the right side of the prepared humanized mouse. Avelumab was prepared according to the method suggested by the manufacturer, and when the tumor size of humanized mice was 200 to 300 mm ³ , the anticancer agent was administered by intraperitoneal injection twice a week (Monday and Thursday) started The administration period was according to the method suggested by the anticancer drug manufacturer. After tumor transplantation, the tumor size was measured using a caliper twice a week.

For tumor size greater than 1500 mm ³ , BW < 20% vs. In the case of max BW, when the walking of the mouse is uncomfortable, it is difficult to consume feed or negative water, or when it falls into an unconscious state and does not respond to external stimuli, it was euthanized using a carbon dioxide euthanasia chamber and the experiment was humanely terminated.

The humanized mice were observed and tested according to the cycle described in the observation section. Statistically, t-tests for mean and standard deviation were performed for all evaluation items per experimental group, and analysis was performed. When the p value was less than 0.05, it was determined that there was a statistically significant difference.

As a result, it was confirmed that the size of the tumor was significantly reduced according to the administration of avelumab. These results mean that the humanized mouse can be usefully utilized to evaluate the efficacy of immune anticancer drugs.

Claims (15)

isolating cells from the biological sample;
Preparing induced pluripotent stem cells (iPSCs) from the isolated cells;
Inducing hematopoietic stem cells (HSC) from the prepared dedifferentiated stem cells; and
A method for producing an animal model, comprising administering the induced hematopoietic stem cells to an animal.
The method according to claim 1, wherein the inducing step comprises the following steps:
(a) culturing the dedifferentiated stem cells in a medium to which bone morphogenetic protein 4 (BMP4) is added;
(b) culturing by replacing the cells cultured in step (a) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added;
(c) culturing the cells cultured in step (b) by replacing them with a medium to which SB431542, vascular endothelial growth factor, and basic fibroblast growth factor are added;
(d) culturing by replacing the cells cultured in step (c) with a medium to which stem cell factor (SCF), thrombopoietin (TPO), and interleukin-3 (IL-3) are added;
(e) obtaining a culture supernatant of the cells cultured in step (d);
(f) separating the cells contained in the obtained supernatant; and
(g) the isolated cells Flt3L (FMS-like tyrosine kinase 3 ligand), stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), and interleukin-6 (IL-6) ) culturing in the added medium.
The method according to claim 1, wherein the administering is to use an animal of 3 to 5 weeks of age.
The method according to claim 1, wherein the administration is an intravenous injection.
The method according to claim 1, wherein the administering is that the hematopoietic stem cells are administered at 1x10 ⁶ cells per animal. Methods for making animal models.
The method according to claim 1, wherein the animal model is any one selected from the group consisting of mouse, rat, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig, and goat.
The method according to claim 6, wherein the animal is an immunodeficient mouse.
The method according to claim 7, wherein the immunodeficient mouse is an NSG mouse.
The method according to claim 1, wherein the isolated cells are mononuclear cells.
The method according to claim 1, wherein the biological sample is derived from a cancer patient.
An animal model for analyzing the therapeutic efficacy of an anticancer agent prepared by the method of claim 1.
The animal model of claim 11 , wherein human CD45 is expressed in at least 45% of leukocytes in blood.
The animal model of claim 11 , wherein the anticancer agent is an immune anticancer agent.
administering an anticancer agent to the animal model of claim 11; and
Analyzing the growth or metastasis of cancer cells in the animal model, comprising the step of determining the therapeutic efficacy of the anticancer agent, the method of analyzing the therapeutic efficacy of an anticancer agent.
administering an anticancer drug candidate to the animal model of claim 11; and
Analyzing the growth or metastasis of cancer cells in the animal model, comprising the step of determining the therapeutic efficacy of the anticancer agent, the screening method of an anticancer agent.

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