KR20220113586A - Method for producing gene-edited cells using pluripotent stem cell-derived hemogenic endothelial cells - Google Patents

Abstract

The present invention relates to a method for manufacturing gene-edited cells using pluripotent stem cell-derived hematopoietic endothelial cells, wherein the gene-edited cells can be obtained stably and with high efficiency by using hematopoietic endothelial cells inducible into lymphoid cells. More specifically, the present invention relates to a method for manufacturing gene-edited hematopoietic endothelial cells comprising the following steps: preparing hematopoietic endothelial cells (HEC); injecting a delivery vehicle for gene editing into the hematopoietic endothelial cells; and obtaining gene-edited hematopoietic endothelial cells.

Description

Method for producing gene-edited cells using pluripotent stem cell-derived hemogenic endothelial cells

It relates to a method for producing a gene-edited cell using pluripotent stem cell-derived hematopoietic endothelial cells.

Although there is a high level of recognition for the stable differentiation technology of hemangioblasts, there are no reports on the differentiation of hemangioblasts and their use as base cells for loading genetically modified products. Currently, gene editing is used as a method for mounting the killing ability of natural killer cells, but the transfection efficiency of primary PB-derived natural killer cells or NK92mi cell lines is very low, so there is a problem that the gene cannot be inserted efficiently. On the other hand, hemangioblasts have the advantage of being able to attach to a culture dish, and thus have high transfection efficiency in the cells, and divide with information about gene editing during cell division. In addition, since hemangioblasts possess the characteristics of progenitor cells, they can be differentiated into lymphoid cells or natural killer cells, and genetic modification can be attempted with high efficiency. Therefore, there is a need to develop hemangioblasts that enable the generation of blood cells such as lymphocytes or natural killer cells as base cells for gene editing.

One aspect is to prepare hematopoietic endothelial cells (Hemogenic endothelial cells, HEC); injecting a carrier for gene editing into the hematopoietic endothelial cells; And to provide a method for producing a gene-edited hematopoietic endothelial cell comprising the step of obtaining a gene-edited hematopoietic endothelial cell.

One aspect is to prepare hematopoietic endothelial cells (Hemogenic endothelial cells, HEC); injecting a carrier for gene editing into the hematopoietic endothelial cells; and obtaining a gene-edited hematopoietic endothelial cell.

As used herein, the term "hemogenic endothelial cell" refers to a rare differentiated vascular endothelial cell, which can be differentiated into a hematopoietic cell (hematoblast) during embryogenesis. The development of hematopoietic cells in the embryo sequentially proceeds from the mesoderm to the vascular endothelium and hematopoietic precursor cells through hemangioblasts. The "angioblast, or vasoformative cell" is a type of endothelial precursor cell derived from bone marrow, and is one of the mesenchymal cells that can develop into the endothelium of blood vessels.

In one embodiment, the hematopoietic endothelial cells are mesodermal cells by culturing pluripotent stem cells in a first medium containing a first medium composition comprising bFGF, VEGF and SCF. differentiating; culturing the mesoderm cells in a second medium containing a second medium composition comprising TPO, EPO and IGF-1 to differentiate them into early hemogenic endothelial cells (EHE); and culturing the EHE in a third medium containing a third medium composition comprising IL-5, IL-7 and DLL to differentiate into late hemogenic endothelial cells (LHE). can The medium composition may be included in a conventional cell culture medium. The cell culture medium is, for example, DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, USA), MEM (Minimal Essential Medium, GIBCO, USA), BME (Basal Medium Eagle, GIBCO, USA), RPMI 1640 (GIBCO, USA). ), DMEM/F10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10; GIBCO, USA), DMEM/F12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12; GIBCO, USA), α-MEM (α-Minimal essential) Medium; GIBCO, USA), G-MEM(Glasgow's Minimal Essential Medium, GIBCO, USA), IMDM(Isocove's Modified Dulbecco's Medium, GIBCO, USA), Apel Ⅱ(STEMdiff APEL 2 Medium), EGM-2(EGM　Endothelial Cell Growth) Medium) and the like. The step of differentiating into mesodermal cells may be performed for 1 to 3 days. For example, the step may be performed for 1 to 3 days, 1 to 2 days, or 2 to 3 days. At this time, when the culture period is less than the above range, there is a problem that the pluripotent stem cells are not sufficiently differentiated into mesoderm cells. There is a problem of differentiation into cells of In addition, the step of differentiating into early hematopoietic endothelial cells may be performed for 6 to 8 days. At this time, when the culture period is less than the above range, there is a problem that the growth and expansion of hematopoietic endothelial cells may be atrophied. There is a reduction problem. In addition, the step of differentiating into late hematopoietic endothelial cells may be performed for 12 to 13 days. In this case, when the differentiation period is less than the above range, there is a problem that the primary hematopoiesis process does not sufficiently occur, which affects the cell generation and frequency of the definitive hematopoiesis process.

As used herein, the term "pluripotent stem cell (pluripotent stem cell)" is pluripotent (pluripotent) or totipotent (totipotent) capable of differentiating into cells of all tissues of an individual having self-renewal ability (self-renewal) means stem cells. The pluripotent stem cells include, for example, embryonic stem cells, induced pluripotent stem cells (iPSCs), and somatic cell nuclear transfer derived stem cells by somatic cell nuclear transfer.

As used herein, the term "lymphoid lineage blood cell" is a lineage cell differentiated from hematopoietic stem cells, mainly involved in adaptive immunity and innate immunity. Lymphocyte lineage cells include, for example, natural killer cells (NK cells), T lymphocytes, and B lymphocytes. Accordingly, the method according to one aspect stably stabilizes gene-edited cells in all lineages by using hematopoietic endothelial cells that can efficiently differentiate into lymphoid lineage cells such as NK cells, T lymphocytes or B lymphocytes of pluripotent stem cells in vitro. can be obtained with

As used herein, the term "gene editing" refers to a nucleic acid molecule (one or more, for example, 1 to 100,000 bp, 1 to 10,000 bp, 1 to 1,000 bp, 1 to 100 bp, 1 to 70 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp or 1 to 10 bp) can be used to alter, and/or restore (correct) gene function by deletion, insertion, substitution, etc. .

The gene editing may be to transfer a gene using a viral gene delivery system or a non-viral delivery agent. The viral gene delivery system is a DNA virus such as adenovirus, adeno-associated virus, herpes simplex virus, or the like or retino virus, lenti virus. It may be an RNA virus such as In addition, the non-viral carrier may be, for example, direct gene injection, electroporation, gene gun, lipid-gene conjugate, polymer-gene conjugate, and the like.

Gene-edited hematopoietic endothelial cells may include cells that have been gene-edited by targeting one or more genes of interest (GOIs). The gene of interest is, for example, B2M-/-, CIITA-/-, HLA-E+, HLA-G+, Chimeric antigen receptor (CAR)-specific monoclonal antibody, CD3z, 4.1.BB, It may be a cancer stem cell marker, CD44, FLT4, or the like. In addition, the gene of interest may be operably linked to a reporter gene. The "operably linked (operably linked)" means that a nucleic acid encoding a nucleic acid expression control sequence and a target protein are functionally linked to perform a general function. The reporter gene is, for example, a fluorescent protein, beta-galactosidase, beta-lactamase, TEV-protease, dihydrofolate reductase), luciferase, Renilla luciferase, Gaussia luciferase, selection marker, surface marker gene, antibiotic resistance protein, etc. can

In one embodiment, the step of obtaining the gene-edited hematopoietic endothelial cells may include selectively killing cells whose expression of a gene of interest is not increased or suppressed by gene editing. The selectively killing the cells may be performed by treating a cell sample containing the gene-edited hematopoietic endothelial cells with an antibiotic or the like. The gene-edited hematopoietic endothelial cells may be cells that are undergoing cell division or have completed cell division, and the cells may be cultured for 2 to 10 passages. The cell death may refer to apoptosis, unless otherwise stated.

Another aspect provides a hematopoietic endothelial cell produced by the method. In one embodiment, it was confirmed that the transfection efficiency of the hematopoietic endothelial cells was similar to or increased as compared to the embryonic stem cells (CHA562), HDF cells, and NK92mi cells as the cell division progressed. Therefore, hematopoietic endothelial cells according to an aspect can replace existing cells with low gene editing efficiency as cell division proceeds while maintaining information on gene editing. In addition, since the hematopoietic endothelial cells can be differentiated into lymphoid cells, various genetic modifications such as vascular cells and lymphatic cells can be attempted using the cells.

The method according to one aspect can stably and efficiently obtain gene-edited cells by using hematopoietic endothelial cells inducible into lymphoid cells.

1 is a photograph showing the characteristics of early and late hematopoietic endothelial cells.
Figure 2a is a photograph showing the cell morphology according to the culture time of the late hematopoietic endothelial cells and a graph showing the proliferation of single cells.
Figure 2b is a photograph confirming the expression of marker protein in late hematopoietic endothelial cells.
Figure 3a is a photograph showing the cell shape after cultured HE in Apel II basal medium 1, 3, 7, and thereafter.
Figure 3b is a photograph showing the cell shape of days 1 and 3 of culturing HE in EGM-2 basal medium.
Figure 3c is a photograph showing the cell shape after culturing HE in DMEM/F12 basal medium on day 1, day 3, day 7, and thereafter.
Figure 4a is the result of confirming the marker protein in the late hematopoietic endothelial cells.
Figure 4b is a photograph confirming the Tie-2 enrichment of CD31 cells.
Figure 5a is the result of confirming the lymphocyte cell generation in hematopoietic endothelial cells in the liver of mice.
5B is a result confirming the generation of lymphocytes in hematopoietic endothelial cells in mouse AGM.
Figure 5c is a chart showing the appearance and colony formation of hematopoietic endothelial cells cultured in each basal medium, and the generation of lymphocytes and myeloid cells, and confirming the shape of hematopoietic endothelial cells.
Figure 5d is a graph showing the results of analyzing the expression levels of CD45, CD4, CD8, NK1.1 using the differentiated cells in AGM.
6A is a result showing the characteristics and expression markers of cells in each stage of generation of lymphoid cells from human pluripotent stem cell-derived hematopoietic endothelial cells.
Figure 6b is a graph confirming whether differentiation into lymphocyte-based cells is made in the spot treated with IL-5 and DDL1 by the expression of the lymphatic transcription factor.
7a is a microscopic image of the bright field and GFP-expressing hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells on the 6th and 9th days in situ.
7B is a graph qualitatively analyzed by FACS analysis of the fluorescence levels of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells.
Figure 7c is a graph quantifying the degree of fluorescence of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells.
Figure 8a induced cell division of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells at an MOI of 10 until day 9, and whether GFP-transfected cells and CFSE were co-expressed at the cell level. This is the result confirmed in Red boxes indicate coexpression.
Figure 8b induced cell division of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells at an MOI of 50 until day 9, and whether GFP-transfected cells and CFSE were co-expressed at the cell level. This is the result confirmed in
8c shows the results of inducing division of hematopoietic endothelial cells (HE) and HDF cells up to day 9 at MOI 10 and MOI 50, and quantifying the expression of GFP and PE-congugated CFSE on days 6 and 9.
Figure 8d induced cell division of hematopoietic endothelial cells (HE), and HDF cells by day 9 at MOI 10 and MOI 50, and quantification of CFSE and dividing cells in each cell expressing GFP on days 6 and 9. It is one graph.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Production Example]

Preparation Example 1. Establishment of culture conditions for differentiation and maintenance of stem cells into hematopoietic endothelial cells

The differentiation and culture conditions of stem cells into hematopoietic endothelial cells were confirmed. Specifically, 2.0 x 10 ⁵ stem cells (hereinafter, 'CHA52' cells) obtained from CHA Biotech Co., Ltd. were dispensed into 1 well coated with Matrigel in a 6 well culture dish and cultured for two days. Then, the cells were incubated in a basal medium (Stemline II medium) for mesoderm containing bFGF 7 ng/ml, CHIR 1 nM, ascorbic acid 60 ng/ml, VEGFA 15 ng/ml, SCF 35 ng/ml and BMP4 18 ng/ml. It was cultured for 3 days in a specific conditioned medium (hereinafter, 'Step I'). Then, the cells were incubated in a basal medium (Apel II medium) with bFGF 7 ng/ml, VEGFA 15 ng/ml, SCF 180 ng/ml, FLT3L 150 ng/ml, GCSF 18 ng/ml, TPO 80 ng/ml, EPO It was further cultured for 6 days in an optimized medium containing 17 ng/ml, 25 ng/ml of IL-6 and 30 ng/ml of IGF-1 (hereinafter, 'Step II'). The period up to the 6th day was termed pro hemogenic endothelial cells (pro HE), and on the 6th day, hematopoietic endothelial cells were subcultured and isolated with high purity. At this time, it was confirmed that non-hematopoietic endothelial cells distributed as a single layer fell first by treatment with 0.25% Trypsin/EDTA in an incubator for 3 minutes. After washing the medium with DPBS, 0.25% Trypsin/EDTA was further treated to separate hematopoietic endothelial cell clusters into single cells. After filtering the isolated single cells with a 0.44 µm filter, divide the cells from 1 well into 2 wells and add bFGF 11 ng/ml, VEGFA 50 ng/ml, SCF 170 ng/ml, FLT3L to basal medium (Apel II medium). 150 ng/ml, GCSF 18 ng/ml, IL-6 35 ng/ml, IGF-1 25 ng/ml, IL-7 15 ng/ml, IL-15 10 ng/ml, IL-5 25 ng/ml and subcultured for 21 days in a conditioned medium containing 7 ng/ml of DLL1 (hereinafter, 'Step III'). A half change of the medium was maintained once every 3 to 4 days until the 21st.

Preparation Examples 2-4.

It was carried out in the same manner as in Preparation Example 1, except that the composition and content of Tables 1 to 3 were included in each step.

composition Preparation 2 Preparation 3 Preparation 4 Preparation 5 Phase I bFGF 15 ng/ml 9 ng/ml 12 ng/ml 17 ng/ml CHIR 5 nM 2 nM 4 nM 7 nM ascorbic acid 70 ng/ml 80 ng/ml 120 ng/ml 150 ng/ml VEGFA 18 ng/ml 22 ng/ml 25 ng/ml 30 ng/ml SCF 55 ng/ml 45 ng/ml 58 ng/ml 65 ng/ml BMP4 20 ng/ml 22 ng/ml 30 ng/ml 35 ng/ml

composition Preparation 2 Preparation 3 Preparation 4 Preparation 5 Phase II bFGF 15 ng/ml 9 ng/ml 12 ng/ml 17 ng/ml VEGFA 18 ng/ml 22 ng/ml 25 ng/ml 30 ng/ml SCF 225 ng/ml 195 ng/ml 210 ng/ml 270 ng/ml FLT3L 170 ng/ml 190 ng/ml 210 ng/ml 230 ng/ml GCSF 25 ng/ml 23 ng/ml 27 ng/ml 30 ng/ml TPO 150 ng/ml 120 ng/ml 130 ng/ml 180 ng/ml EPO 23 ng/ml 21 ng/ml 25 ng/ml 27 ng/ml IL-6 35 ng/ml 45 ng/ml 55 ng/ml 65 ng/ml IGF-1 40 ng/ml 35 ng/ml 45 ng/ml 55 ng/ml

composition Preparation 2 Preparation 3 Preparation 4 Preparation 5 Phase III bFGF 13 ng/ml 15 ng/ml 20 ng/ml 23 ng/ml VEGFA 30 ng/ml 70 ng/ml 90 ng/ml 110 ng/ml SCF 230 ng/ml 200 ng/ml 300 ng/ml 330 ng/ml FLT3L 180 ng/ml 210 ng/ml 240 ng/ml 270 ng/ml GCSF 25 ng/ml 22 ng/ml 28 ng/ml 32 ng/ml IL-6 45 ng/ml 40 ng/ml 55 ng/ml 65 ng/ml IGF-1 40 ng/ml 30 ng/ml 50 ng/ml 55 ng/ml IL-7 25 ng/ml 18 ng/ml 28 ng/ml 35 ng/ml IL-15 23 ng/ml 17 ng/ml 30 ng/ml 37 ng/ml IL-5 40 ng/ml 30 ng/ml 50 ng/ml 65 ng/ml DDL1 35 ng/ml 15 ng/ml 28 ng/ml 30 ng/ml

[Example]

Example 1. Confirmation of differentiation of stem cells into hematopoietic endothelial cells

1-1. Characterization of hematopoietic endothelial cells

After dividing the hematopoietic endothelial cells cultured in Step III of Preparation Example 1 into early hemogenic endothelial cells (EHE) and late hematopoietic endothelial cells (LHE) on the 13th day of culture, The appearance of each cell was confirmed using a microscope (x 100, x 200 and x 400).

1 is a photograph showing the characteristics of early and late hematopoietic endothelial cells.

As a result, as shown in FIG. 1 , in the case of early hematopoietic endothelial cells, it was confirmed that clusters were formed with a non-wide distribution of cell masses, and it was confirmed that cells other than the initial hematopoietic endothelial cells were distributed together. At this time, it was confirmed that marker proteins of hematopoietic endothelial cells such as CD31 and VE-Cadherin were expressed.

In addition, in the case of the late hematopoietic endothelial cells after the 13th day of culture, it was confirmed that more cells were aggregated than in the early hematopoietic endothelial cells, and the emerging blood cells showed the shape of definitive blood. It was confirmed that these late hematopoietic endothelial cells reached the end of their lifespan as the cytoplasm became thinner and thinner as the culture progressed. In addition, it was confirmed that FLK-1+ megakaryocytes-red blood cell progenitors (Progenitors) emerged from the late hematopoietic endothelial cells. As a result of clonal expansion by generating several megakaryocyte-erythrocyte progenitors from one of these late hematopoietic endothelial cells, the hematopoietic endothelial cells mainly show rod-like cells, which are typical of cobblestone-shaped endothelial cells. -like cells) or mesenchymal stem cells (mesenchymal stromal cells) were found to be different in shape. The blood cells formed by the late hematopoietic endothelial cells are mainly primitive blood cells (mainly myeloid) in the early stage, and definitive blood cells (including both myeloid and lymphocytes) in the late hematopoietic endothelial cell-derived cells. It was confirmed that tube formation was formed in the generated vascular endothelial cells. Judging from the above results, it is considered that hematopoietic endothelial cells possess both the characteristics of vascular endothelial cells and those of blood cells.

Figure 2a is a photograph showing the cell morphology according to the culture time of the late hematopoietic endothelial cells and a graph showing the proliferation of single cells.

As a result, as shown in FIG. 2A , on the first day of culture of late hematopoietic endothelial cells, the porphyrin precursor, 5-aminolevulinate synthase, was exposed to bright light presumed to be caused by the enzyme. (indicated by a white arrow) was confirmed to show bright yellow light under the microscope. However, it was confirmed that the bright light disappears from the hematopoietic endothelial cells after the generation of erythroblasts (D9, after the 10th day). In addition, in the hematopoietic endothelial cells on the 5th day, the shape of budding erythroblasts could be confirmed, and it was confirmed that the erythroblasts were formed after the 7th day. It was confirmed that the doubling day of the hematopoietic endothelial cells was about 1.2 days, and the average doubling time was about 29.5 hours.

2b is a photograph confirming the expression of marker protein in late hematopoietic endothelial cells.

As a result, as shown in Figure 2b, it was confirmed that the marker proteins CD31, Tie-2, CD34, etc. were expressed in the hematopoietic endothelial cells forming the cluster after the 9th day, and in some cells, mesodermal markers such as RUNX1 and brachuary were expressed. It was possible to confirm what remained in the nucleus. In addition, it was confirmed that the FLK-1 protein was intensively expressed in the blood cells when they emerged from the hematopoietic endothelial cells to the blood cells.

1-2. Proliferation and differentiation of late hematopoietic endothelial cells according to culture conditions

The late hematopoietic endothelial cells cultured in Example 1-1 were stored frozen for 1 month, and then thawed in a water bath at 37°C. Thereafter, three basic media (Apel II, EGM-2, and DMEM/F12 media) were used to identify media suitable for proliferation and differentiation of late hematopoietic endothelial cells, and in the case of Apel II and DMEM/F12 media, 7 ng of bFGF /ml, VEGFA 15ng/ml, SCF 180ng/ml, FLT3L 150ng/ml, GCSF 18ng/ml, TPO 80ng/ml, EPO 17ng/ml, IL-6 25ng/ml, IGF-1 30 ng/ml, IL-3 45 ng/ml and CHIR 1 nM were added. In the case of EGM-2 medium, only the cytokine Lonza (cat no CC-3202) was additionally added. Thereafter, the frozen and thawed late hematopoietic endothelial cells were cultured in each of the three media, and all media maintained a half change of the media once every 3 to 4 days. In the case of late hematopoietic endothelial cells, since they are vascular endothelial cells thawed after freezing, it was expected that they would attach to the culture dish and proliferate.

Figure 3a is a photograph showing the cell shape after cultured hematopoietic endothelial cells in Apel II basal medium 1, 3, 7, and thereafter.

As a result, as shown in FIG. 3a , it was confirmed that most of the hematopoietic endothelial cells failed to attach to the culture dish and quickly died in the Apel II basal medium. However, in the case of hematopoietic endothelial cells that survived after 7 days, it was confirmed that when their function as endothelial cells was stabilized, WBC-like cells were generated or erythroblast colonies were formed. However, it was significantly lower than the blood cell production of hematopoietic endothelial cells cultured in EGM-2 and DMEM/F12 basal medium.

Figure 3b is a photograph showing the cell shape on day 1 and day 3 of culturing hematopoietic endothelial cells in EGM-2 basal medium.

As a result, as shown in FIG. 3b , hematopoietic endothelial cells cultured in EGM-2 basal medium proliferated stably after attachment to the culture dish, and their proliferation rate was also faster and more stable than other basal media. In particular, in the case of hematopoietic endothelial cells proliferated for more than 7 days, it was confirmed that they were divided into a typical cobblestone-shaped endothelial progenitor cell and an atypical vascular endothelial cell-shaped cell group. On the other hand, among the cells cultured in Apel II basal medium, non-adherent cells were cultured in EGM-2 medium for 3 days to induce proliferation of adherent cells, and then cultured in Apel II medium again. As a result, hematopoietic endothelial cells was found to proliferate normally. That is, the EGM-2 basal medium is positive for proliferation and engraftment of hematopoietic endothelial cells, and can affect the production of blood cells by stably attaching and proliferating hematopoietic endothelial cells in a culture dish.

Figure 3c is a photograph showing the cell shape on day 1, day 3, day 7 and thereafter after culturing hematopoietic endothelial cells in DMEM/F12 basal medium.

As a result, as shown in FIG. 3C , it was confirmed that hematopoietic endothelial cells cultured in DMEM/F12 basal medium adhered to the culture dish and proliferated. Compared with hematopoietic endothelial cells cultured in EGM-2 basal medium, the proliferation rate was delayed by 4 to 5 days, but many colonies of hematopoietic endothelial cells were formed, and it was confirmed that a large amount of blood cells were stably generated after 8 days. .

1-3. Identification of protein markers expressed in hematopoietic endothelial cells

The expression of marker proteins expressed in hematopoietic endothelial cells according to the basal medium of Example 1-2 was compared. As a result, the expression of marker proteins according to the basal medium was not significantly different, and it was confirmed that typical marker proteins were expressed in hematopoietic endothelial cells.

Figure 4a is the result of confirming the marker protein in hematopoietic endothelial cells cultured in EGM-2 basal medium for 3 days.

Figure 4b is a photograph confirming the Tie-2 enrichment of hematopoietic endothelial cells cultured for 14 days in EGM-2 basal medium.

As a result, as shown in Figure 4a, in the hematopoietic endothelial cell group cultured in EGM-2 basal medium, CD31 27.9%, CD34 8.2%, CD144 29.9%, and Flk-1 4.5% protein markers were expressed, and CD144+CD31+ It was confirmed that 11.3%, CD144+CD34+ 8.5%, CD31+CD34+ 10.2%, and Flk-1+CD34+ 4.9% protein markers were expressed. In addition, as shown in FIG. 4b , it was confirmed that the expression of Tie-2 was 24.9% enriched in the CD31+ cell group in the MACS sorting group using CD31, and 1.8% enriched in the CD31- cell group. These results suggest the commitment of CD31+ cells to hematopoietic endothelial cells. In addition, it was confirmed that hematopoietic endothelial cell markers were expressed in the order of Tie2 > CD144 > CD31 > CD34 > FLK1.

1-4. Confirmation of culture conditions of hematopoietic endothelial cells for induction of lymphoid cell production

Based on the results of Example 1-2, culture conditions for hematopoietic endothelial cells for inducing lymphocyte production were additionally confirmed. First, Arota gonad mesonephros (AGM) and liver tissue were isolated from rat fetus at 10.5 to 11.5 days of age. Thereafter, the tissue minced behind the lid of the sterilized 1.5 ㎖ eppendorf tube was washed with DPBS, and then cultured in the same three mediums as in Example 1-3 until the 20th day, the birth date of mice. Hematoxylin & Eosin staining was performed to confirm the shape of hematopoietic endothelial cells cultured in each medium.

Figure 5a is the result of confirming the generation of lymphocytes in hematopoietic endothelial cells in the liver of a mouse, Figure 5b is the result of confirming the generation of lymphocytes in the hematopoietic endothelial cells in the mouse AGM. Figure 5c is a chart showing the appearance and colony formation of hematopoietic endothelial cells cultured in each basal medium, and the production degree of lymphoid cells and myeloid cells, and confirming the shape of hematopoietic endothelial cells.

Although hematopoietic endothelial cells were hardly obtained from liver tissue-derived blood cells, it was confirmed that blood cells produced from hematopoietic endothelial cells showed different trends according to each of the three media in the process of infiltration and maturation into the liver. Specifically, as shown in FIG. 5a , in the case of EGM-2 basal medium, it was confirmed that all hematopoietic endothelial cells were killed. On the other hand, in the case of Apel II basal medium, cells were rapidly induced from megakaryocytes-red blood cells and pronormoblasts to orthochromic normoblasts within 3 days, and it was confirmed that the cells disappeared within 6 days. In addition, in the case of DMEM/F12 basal medium, the differentiation of megakaryocytes-erythrocyte-derived megakaryocytes and megakaryocytes (MK-II) rather than erythrocytes was vigorous, and the formation of proplatelet protrusions and silia was remarkably confirmed. In particular, it was confirmed that the production of leukocyte-based cells was greater than that of heme-mounted erythrocyte-type cells.

On the other hand, as shown in FIG. 5b , in the case of hematopoietic endothelial cells present in AGM, it was confirmed that the culture level was different up to the 9th day depending on the medium. Specifically, in the case of the EGM-2 basal medium, it was confirmed that the annihilation of blood cells increased remarkably. That is, it can be seen that, unlike human pluripotency-derived hematopoietic endothelial cells, mouse hematopoietic endothelial cells do not survive for a long period of time. However, in the case of Apel II basal medium, it was confirmed that the enrichment of the red blood cells was made rapidly and the proliferation of the white blood cells was significantly increased. In addition, in the case of DMEM/F12 basal medium, it was confirmed that the degree of differentiation of megakaryocytes and leukocytes was significantly higher than that of erythroid cells. As a result, as shown in Fig. 5c, liver and AGM-derived cells exhibited active adhesion and cell proliferation in DMEM/F12 and Apel II basal media compared to EGM-2 basal media, enabling long-term differentiation into hematopoietic endothelial cells and hematopoietic cells. can be found

Figure 5d is a graph showing the results of analyzing the expression levels of CD45, CD4, CD8, NK1.1 using the differentiated cells in AGM.

As a result, as shown in FIG. 5d , in the case of Apel II basal medium, the induction of natural killer cells in floating cells was the highest on day 10, and in the case of DMEM/F12 basal medium, CD4 and CD8 cells along with megakaryocytes on day 6 were the highest. It was confirmed that the induction rapidly increased and maintained. Accordingly, it can be seen that AGM-derived blood cells are maintained in both of the two basal media.

1-5. Confirmation of differentiation of human pluripotent stem cells into hematopoietic endothelial cells and blood cells

Human pluripotent stem cells were differentiated into hematopoietic endothelial cells and blood cells in the medium of Example 1-2 based on the confirmation that the hematopoietic endothelial cells derived from the mouse fetus were differentiated into blood cells in Example 1-4. It was checked whether or not Specifically, hematopoietic endothelial cells cultured for 21 days according to steps I to III in Preparation Example 1 were further cultured until the 34th day. At this time, after 28 days of culture, the functional window of hematopoietic endothelial cells was set as 21 to 28 days because the hematopoietic endothelial cells were eliminated or the frequency of cell death increased. Only hematopoietic endothelial cells cultured until the 34th day were further treated with 0.25% Trypsin/EDTA to separate cell clusters into single cells. Thereafter, CFU-assay was performed to confirm whether colony formation of myeloid cells was performed.

6A is a result showing the characteristics and expression markers of cells at each stage of generation of lymphocytes from hematopoietic endothelial cells derived from human pluripotent stem cells.

FIG. 6b is a graph confirming whether differentiation into lymphocyte-based cells occurs in a spot treated with IL-5 and DDL1 by expression of a lymphoblastic transcription factor.

As a result, as shown in FIG. 6a , it was confirmed that the hematopoietic endothelial cells cultured from day 13 to day 34 produced colonies of myeloid cells and differentiated. In addition, as shown in FIG. 6b , it was confirmed that a large number of lymphocyte-based transcription factors were expressed in spots treated with IL-5, DLL1, and the like. That is, it can be seen that the medium according to one aspect is capable of long-term culture of human pluripotent stem cells, so that differentiation into hematopoietic endothelial cells and vascular cells (lymphocytes) is possible.

Example 2. Confirmation of transfection efficiency of hematopoietic endothelial cells

Looking at the current trend of attempting gene editing using pluripotent stem cells (PSC), master cell banking attempts in the state of gene editing are starting, which is important in the cell therapy market. is suggesting However, there are technical limitations because only the process of verifying cell proliferation in a single cloned cell and the process of editing one gene at a time are allowed. On the other hand, another alternative for generating gene-edited NK cells is to use primary PB NK cells and the NK92mi cell line using allogenic NK cells as a starting point. However, the transfection efficiency of these cells is not as high as that of T cells due to the innate property that these cells may also be resistant to the virus to be transfected. In general, fibroblasts such as 293T cells or HDF cells are used for transfection. In particular, in the case of immortalized cell line cells such as 293T cells, since they have the property of cell division to the end, they are widely used for transfection. There is a limitation in using it after direct differentiation into CAR-NK, etc. However, hematopoietic endothelial cells have strong progenitor cell properties, so they can have the strength of gene editing and maintain high efficiency, so they can be used as CAR-NK and Universal NK.

Therefore, based on the above background, the possibility of using the hematopoietic endothelial cells for which the culture conditions were established in Example 1 as a base cell for gene editing was confirmed. After the GFP-expressing construct was inserted into the hematopoietic endothelial cells of Example 1 using a lentivirus, the cells were cultured. The frequency of GFP-bearing cells was investigated on days 3, 6 and 9 after culture. Specifically, 5 μg pRSV-Rev plasmid, 5 μg pCMV-VSV-G plasmid, 5 μg pCgpV plasmid and 15 μg pSIH1-H1-siLUC-copGFP plasmid were added to 6 mil HEK-293T cells with lipofectamine 2000 (Invitrogen, cat no TFS). -11668019) was introduced and cultured for 72 hours to produce GFP-attached lentivirus. Thereafter, the lentivirus was filtered through a 0.45 μm filter and concentrated at 4° C. for 24 hours using Retro-Concentin Retroviral Concentration Reagent (System Biosciences, cat no RV100A-1). After concentration, the titer of the lentivirus was measured using the QuickTiter Lentivirus Titer Kit (Cellbiolabs, cat no VPK-107), and the measured lentivirus was applied to the hematopoietic endothelial cells of Example 1 at an MOI (multiplicity of infection) 10 or 50. Thereafter, the cells were left for 3 days without changing the medium, and then divided into a FACS analysis group and a continuous culture group on the third day. After culturing the FACS analysis group and the continuous culture group, respectively, the degree of decrease in GFP was measured on the 6th and 9th days. Embryonic stem cells (CHA562), HDF cells, and NK92mi cells were used as controls, and the same method as above was used.

7a is a microscopic image of the bright field and GFP-expressing hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells on the 6th and 9th days in situ.

As a result, as shown in FIG. 7a , it was confirmed that the transfection efficiency in embryonic stem cells was the lowest at MOI 10 and MOI 50 . In addition, in the case of NK92mi cells, GFP-expressing cells remained to some extent at MOI 50, but did not show sophisticated fluorescence like HDF cells or hematopoietic endothelial cells.

7B is a graph qualitatively analyzed by FACS analysis of the fluorescence levels of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells.

As a result, as shown in FIG. 7b , it was confirmed that all cells exhibited a stronger degree of fluorescence at MOI 50 compared to MOI 10 . On the other hand, in the case of NK92mi cells, unlike observed under a microscope (see FIG. 7a ), it was confirmed that GFP-expressing cells were not enriched in cell scattering.

Figure 7c is a graph quantifying the degree of fluorescence of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells.

As a result, as shown in FIG. 7c , it was confirmed that the initial (3rd day) transfection efficiency of embryonic stem cells was the lowest regardless of the MOI. In addition, in the case of NK92mi cells, the transfection efficiency was about 10% at MOI 50 (day 3), but it was confirmed that the frequency of GFP-expressing cells decreased significantly over time. In addition, HDF cells increased until the 6th day regardless of the MOI, and in the case of the MOI50, the transfection efficiency was about 20% on the 6th day, but it was confirmed that it decreased significantly after the 6th day. On the other hand, in the case of hematopoietic endothelial cells, the transfection efficiency was the highest on day 6 regardless of the MOI and showed a decreasing trend thereafter, but showed the highest efficiency compared to embryonic stem cells, HDF cells and NK92mi cells (day 9). ). That is, hematopoietic endothelial cells according to an aspect can be used as base cells for transfection by stably delivering information about gene editing to passaged cells despite cell division progressing.

Example 3. Confirmation of transfection stability of hematopoietic endothelial cells

In order to confirm the transfection stability of hematopoietic endothelial cells into which the GFP construct was inserted in Example 2, GFP expressed in hematopoietic endothelial cells co-expressed with CFSE (proliferation marker) was quantified. Specifically, 1.0 x 10 ⁶ hematopoietic endothelial cells of Example 2 were placed in a beaker containing 1 ml of DPBS, and 1 μl of PE-conjugated CFSE (Carboxylfluorescein Diacetate Succinimidyl Ester) (Invitrogen, Cat No C34573) stock was added thereto. Exposure at room temperature for 20 minutes. After that, the culture solution was filled so that the total volume of the beaker was 5 ml, washed and further cultured to investigate the frequency of GFP-bearing cells on days 3, 6, and 9, and cells undergoing cell division were subjected to FACS. It was analyzed and quantified. Embryonic stem cells (CHA562), HDF cells, and NK92mi cells were used as controls, and the same method as above was used.

Figure 8a induced cell division of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells at an MOI of 10 until day 9, and whether GFP-transfected cells and CFSE were co-expressed at the cell level. This is the result confirmed in Red boxes indicate coexpression.

As a result, as shown in Figure 8a, in the case of embryonic stem cells, it was confirmed that cell division was successful in some cells, and in the case of NK92mi cells, a large number of cells were divided. However, it was confirmed that the transfection failed because no GFP-expressing cells were observed in the cells. On the other hand, in the case of HDF cells and hematopoietic endothelial cells, it was confirmed that cell division and transfection were successful in some cells.

Figure 8b induced cell division of hematopoietic endothelial cells (HE), embryonic stem cells (CHA562), HDF cells and NK92mi cells at an MOI of 50 until day 9, and whether GFP-transfected cells and CFSE were co-expressed at the cell level. This is the result confirmed in

As a result, as shown in FIG. 8b , it was confirmed that the MOI 50 also exhibited a similar aspect to the MOI 10 (see FIG. 8a ). However, at MOI 50, the intensity of GFP fluorescence is stronger in HDF cells and hematopoietic endothelial cells, confirming that the frequency of transfected cells is higher.

8c shows the results of inducing division of hematopoietic endothelial cells (HE) and HDF cells up to day 9 at MOI 10 and MOI 50, and quantifying the expression of GFP and PE-congugated CFSE on days 6 and 9.

As a result, as shown in FIG. 8c , the presence of double color was confirmed in hematopoietic endothelial cells and HDF cells, and it was confirmed that the intensity of CFSE decreased while the cells were dividing.

Figure 8d induced cell division of hematopoietic endothelial cells (HE), and HDF cells by day 9 at MOI 10 and MOI 50, and quantification of CFSE and dividing cells in each cell expressing GFP on days 6 and 9. It is one graph.

As a result, as shown in FIG. 8d , in the case of HDF cells, cells that successfully transfected showed a frequency of about 12% among cells undergoing cell division regardless of MOI (day 6), but on the 9th day, It was confirmed that the frequency decreased significantly. On the other hand, in the case of hematopoietic endothelial cells, the initial efficiency of transfection and cell division was lower than that of HDF cells, but it was confirmed that the efficiency was maintained or slightly increased until the 9th day. That is, hematopoietic endothelial cells according to an aspect can maintain gene editing efficiency even during cell division, and thus can be utilized as base cells for transfection.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (10)

Preparing hematopoietic endothelial cells (HEC);
injecting a carrier for gene editing into the hematopoietic endothelial cells; and
A method for producing a gene-edited hematopoietic endothelial cell comprising the step of obtaining a gene-edited hematopoietic endothelial cell. The method according to claim 1, wherein the gene editing is to deliver the gene using a viral gene delivery system or a non-viral delivery agent. The method according to claim 2, wherein the viral gene carrier is adenovirus (adeno virus), adeno-associated virus (adeno-associated virus), herpes simplex virus (Herpes simplex virus), retino virus (retino virus), lenti virus (lenti virus) ) is selected from the group consisting of. The method according to claim 2, wherein the non-viral carrier is selected from the group consisting of direct injection of a gene, electroporation, a gene gun, a lipid-gene conjugate, and a polymer-gene conjugate. The method according to claim 1, wherein the gene-edited hematopoietic endothelial cells include cells that have been gene-edited by targeting one or more genes of interest (GOI). The method of claim 5 , wherein the gene of interest is operably linked to a reporter gene. The method according to claim 6, wherein the reporter gene is a fluorescent protein, beta-galactosidase (beta-galactosidase), beta-lactamase (β-lactamase), TEV- protease (TEV-protease), dihydrofolate reductase ( Dihydrofolate reductase), luciferase (luciferase), Renilla luciferase (Renilla luciferase), Gaussia luciferase (Gaussia luciferase), selection marker (selection marker), surface marker gene (surface marker gene) and antibiotic resistance protein and encoding a protein selected from the group consisting of. The method according to claim 1, wherein the step of obtaining the gene-edited hematopoietic endothelial cells comprises the step of selectively killing cells in which the expression of the gene of interest is not increased or suppressed by gene editing. The method according to claim 1, wherein the hematopoietic endothelial cells are differentiated from pluripotent stem cells. The method according to claim 9, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells (iPSCs), and somatic cell nuclear transfer derived stem cells. .

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