METHOD FOR PREPARING PLURIPOTENT STEM CELL-DERIVED HEMATOPOIETIC STEM CELL AND METHOD FOR CONSTRUCTING HUMANIZED MOUSE MODEL BY USING HEMATOPOIETIC STEM CELL THUS PREPARED

Abstract

A method for preparing pluripotent stem cell-derived hematopoietic stem cells and a method of constructing a humanized mouse model with the prepared hematopoietic stem cells. The method of preparing the hematopoietic stem cells identified an optimal differentiation condition according to a combination of low-molecular-weight compounds and protein growth factors without gene insertion, and thus may differentiate hematopoietic stem cells from pluripotent stem cells at high yield.

Claims

1. A method of preparing hematopoietic stem cells, comprising: culturing pluripotent stem cells (PSCs) in a medium comprising a glycogen synthase kinase (GSK) inhibitor to obtain mesodermal cells; culturing the mesodermal cells to induce or differentiate the mesodermal cells into hemangioblasts; culturing the hemangioblasts to obtain Endothelial-to-Hematopoietic Transition (EHT)-induced hemangioblasts; and culturing the EHT-induced hemangioblasts to induce or differentiate the EHT-induced hemangioblasts into the hematopoietic stem cells.

2. The method of claim 1, wherein the glycogen synthase (GSK) inhibitor is CHIR99021.

3. The method of claim 1, wherein the culturing of the mesodermal cells comprises: culturing the mesodermal cells in a medium comprising at least one growth factor selected from the group consisting of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

4. The method of claim 1, wherein the culturing of the hemangioblasts comprises: culturing the hemangioblasts in a medium comprising a TGF-beta inhibitor.

5. The method of claim 4, wherein the TGF-beta inhibitor is SB-431542.

6. The method of claim 4, wherein the medium for culturing the hemangioblasts further comprises: a retinoic acid (RA) or a pharmaceutically acceptable salt thereof.

7. The method of claim 4, wherein the medium for culturing the hemangioblasts further comprises: at least one growth factor selected from the group consisting of VEGF and bFGF.

8. The method of claim 1, wherein the culturing of the EHT-induced hemangioblasts comprises: culturing the EHT-induced hemangioblasts in a medium comprising a polyvinyl alcohol (PVA) or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the culturing of the EHT-induced hemanagioblasts further comprises: culturing the EHT-induced hemangioblasts in a medium further comprising at least one growth factor selected from the group consisting of stem cell factor (SCF) and bFGF.

10. A method of constructing a humanized animal model, comprising: transplanting or injecting the hematopoietic stem cell prepared by the method of claim 1 into an individual other than a human.

11. A humanized animal model produced by the method of of claim 10.

Description

DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is a diagram schematically showing a novel pluripotent stem cell-derived hematopoietic stem cell differentiation protocol of the present disclosure.

[0063] FIG. 2 is a diagram specifically describing a pluripotent stem cell-derived hematopoietic stem cell differentiation protocol of the present disclosure.

[0064] FIG. 3 is a diagram showing the results of a comparative experiment to establish the primary conditions of inducing hematopoietic stem cells.

[0065] FIG. 4 is a diagram showing the results of a comparative experiment to establish the conditions mesoderm induction.

[0066] FIG. 5 is a diagram showing the results of a comparative experiment to establish the conditions of inducing EHT.

[0067] FIG. 6 is a diagram showing the results of a comparative experiment to establish the secondary conditions of inducing hematopoietic stem cells.

[0068] FIG. 7 is a diagram showing a process of constructing a humanized mouse model by using hematopoietic stem cells prepared by a method of the present disclosure.

[0069] FIG. 8 is a diagram schematically showing a method of preparing hematopoietic stem cells and a method of constructing a humanized mouse model by using the same. of the present disclosure.

MODE FOR INVENTION

[0070] Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present disclosure is not limited to these examples.

Example 1: Novel Human Pluripotent Stem Cell-Derived Hematopoietic Stem Cell Differentiation Protocol

[0071] The present disclosure relates to a method of differentiating human pluripotent stem cells to prepare human hematopoietic stem cells, and a method of constructing a humanized mice by using hematopoietic stem cells prepared by the method. The method may be composed of 1) mesoderm induction/differentiation, 2) hemangioblast induction/differentiation, 3) induction of endothelial-to-hematopoietic transition (EHT), and 4) hematopoietic stem cell (HSC) induction/differentiation, and in each step, treatment with essential low-molecular-weight compounds and/or growth factors (FIG. 1) may be carried out.

[0072] Specific experiments on the method of preparing human hematopoietic stem cells were performed as follows. First, isolated human pluripotent stem cells (hPSCs) were maintained in stemMACS-IPS brew medium for 2 days. The differentiation base culture medium consisted of the following composition: Stempro 34-SFM (+stempro sup)+200 ug/ml of human Transferrin +2 mM of L-glutamine+0.5 mM of L-ascorbic acid+0.45 mM of MTG (1-thioglycerol) +1% of penicillin/streptomycin. To induce mesodermfrom the isolated hPSCs, treatment was performed with CHIR99021 (5 uM) alone, a representative example of a GSK inhibitor, for about 2 days. Next, treatment was performed with 50 ng/ml of BMP4, 50 ng/ml of VEGF, and 100 ng/ml of bFGF for 2 days to induce hemangioblasts. Next, 50 ng/ml of VEGF, 50 ng/ml of bFGF, and SB-431542 (10 M) as representative examples of TGF-beta inhibitors and 1 M of retinoic acid (RA) were added and cultured for about 1 day, and 0.1% (w/v) of PVA, 50 ng/ml of stem cell factor (SCF), and 10 ng/ml of bFGF were added and cultured for about 11 days (FIG. 2).

[0073] In most of the previously published technologies related to hematopoietic stem cell differentiation, differentiation was carried out by forming a colony from hPSCs to Embryonic bodies (EBs), breaking up the colony into single cells, and extracting CD34.sup.+ HSCs, and gene insertion was attempted to increase differentiation efficiency. However, the HSC differentiation method of the present disclosure has the excellent effect of being able to differentiate HSCs with high efficiency and easily obtain HSCs only with an optimal combination of low-molecular-weight compounds and protein growth factors, without the complicated processes mentioned above and without gene insertion.

[0074] Hereinafter, in order to develop a novel hematopoietic stem cell differentiation method of the present disclosure, experiments were performed to establish optimal conditions while comparing with previously published differentiation protocols.

Example 2: Establishment of Primary Conditions of Inducing Hematopoietic Stem Cells

[0075] To establish the primary conditions of inducing hematopoietic stem cells, the following experiment was performed.

[0076] Specifically, it is widely known that vascular endothelial growth factor (VEGF) may be used to induce mesoderm and induce and maintain hemangioblasts (precursors of HSCs), and has been mainly used during the period of inducing hematopoietic stem cells. Therefore, to determine what effect it will have on the final hematopoietic stem cell generation efficiency when VEGF is excluded from the induction of hematopoietic stem cells, the results of hematopoietic stem cell differentiation according to the presence or absence of VEGF (10 ng/ml) were confirmed under the same conditions (50 ng/ml of SCF, 10 ng/ml of bFGF, 20 ng/ml of IL-3 and 10 ng/ml of IL-6) (FIG. 3A).

[0077] To confirm the differentiation results, Fluorescence Activated Cell Sorting (FACS) was performed to confirm the proportion of CD34.sup.+CD45.sup.+ cells. Specifically, to identify cells expressing both CD34 and CD45, anti-CD34 and anti-CD45 antibodies linked with the fluorescent substances PE and PerCP/Cy5.5, respectively, were diluted in staining buffer (phosphate buffered saline (PBS) containing 1% FBS) at a ratio of 1:25. Then, the cells were stained with a cocktail containing the antibodies for 35 minutes at 4 C. After washing away the remaining antibodies with staining buffer, 200 l of staining buffer was added again and analyzed using a flow cytometer.

[0078] As a result, it was confirmed that the ratio of CD34.sup.+CD45.sup.+ cells in the case of excluding VEGF from the induction of hematopoietic stem cells was significantly superior compared to the case in which VEGF was included (FIG. 3B). Based on the above results, it may be seen that, unlike what was previously known, excluding VEGF from the induction of hematopoietic stem cells has a remarkably excellent hematopoietic stem cell differentiation effect.

Example 3: Establishment of Conditions of Inducing Mesoderm

[0079] To establish the conditions of inducing mesoderm (mesodermalcells/mesodermalstem cells), the following experiment was performed.

[0080] Specifically, in the existing method, treatment was performed with bone morphogenetic protein 4 (BMP4), VEGF, and CHIR99021 to induce mesoderm. However, it was determined that the above conditions were not compatible with the sequence of the process for differentiating stem cells into hematopoietic stem cells. Therefore, the hematopoietic stem cell differentiation efficiency was confirmed when treating only with CHIR99021 as a representative example of a GSK inhibitor in inducing mesoderm, excluding BMP4 (5 ng/ml) and VEGF (50 ng/ml), which were previously used for treatment (FIG. 4A).

[0081] As a result, it was confirmed that the ratio of CD34.sup.+CD45.sup.+ cells when treated with CHIR99021 alone in inducing mesodermwas significantly superior compared to when treated with BMP4, VEGF, and CHIR99021 (FIG. 4B). Based on the above results, it may be seen that not treating with BMP4 and VEGF in inducing mesodermhas a remarkably excellent hematopoietic stem cell differentiation effect.

Example 4: Establishment of Conditions of Inducing EHT

[0082] To establish the conditions of inducing endothelial-to-hematopoietic transition (EHT), the following experiment was performed.

[0083] Specifically, in order to induce CD34.sup.+ HSCs, hemangioblasts must be induced after inducing the mesoderm. Since hemangioblasts may be differentiated into CD34.sup.+ HSCs by inducing endothelial-to-hematopoietic transition (EHT), it is important to establish appropriate culture conditions to maximize the efficiency of EHT.

[0084] For this purpose, the efficiency of hematopoietic stem cell differentiation when treated with SB-431542 (SB4), a representative example of a TGF-beta inhibitor, in addition to VEGF and basic fibroblast growth factor (bFGF) for various periods of time, was determined (FIG. 5A). As a result, it was confirmed that when treated with SB4 for less than 24 hours, the proportion of CD34.sup.+CD45.sup.+ cells was significantly superior, and when treated with SB4 for a long period of time, the hematopoietic stem cell differentiation efficiency was significantly reduced (FIG. 5B). In addition, when treated simultaneously with SB4 and retinoic acid (RA), it was confirmed that the hematopoietic stem cell differentiation efficiency was significantly increased compared to when treated with SB4 alone (FIGS. 5C and 5D).

[0085] Based on the above results, it may be seen that inducing EHT after inducing hemangioblasts has a significantly better hematopoietic stem cell differentiation effect when treated with SB4 for about 24 hours, and the combination of SB4 and retinoic acid shows a better hematopoietic stem cell differentiation effect.

Example 5: Establishment of Secondary Conditions of Inducing Hematopoietic Stem Cells

[0086] To establish the secondary conditions of inducing hematopoietic stem cells, the following experiment was performed.

[0087] Specifically, IL-3 and IL-6 are mainly used in the culture process to maintain the CD34.sup.30 potential of hematopoietic stem cells differentiated from human pluripotent stem cells, but this may lead to aging and differentiation of differentiated hematopoietic stem cells, which may slightly reduce the efficiency of maintaining CD34.sup.+ potential. Therefore, the efficiency of maintaining hematopoietic stem cell differentiation was determined when treating with polyvinyl alcohol (PVA) instead of IL-3 and IL-6, which were previously used for treatment, in inducing and maintaining hematopoietic stem cells (FIG. 6A).

[0088] As a result, it was confirmed that when treated with PVA while excluding IL-3 and IL-6 from induction of hematopoietic stem cells, not only did the hematopoietic stem cell differentiation efficiency increase, but the maintenance efficiency of CD34.sup.+ hematopoietic stem cells after differentiation was also significantly superior (FIGS. 6B and 6C). Based on the above results, unlike what was previously known, it may be seen that treating with PVA while excluding IL-3 and IL-6 in inducing hematopoietic stem cells, has a significantly superior hematopoietic stem cell differentiation and maintenance effect.

Example 6: Construction of Humanized Mice

[0089] Based on the experimental results of Examples 2 to 5, the novel hematopoietic stem cell differentiation protocol of Example 1 was established, and humanized mice were constructed using hematopoietic stem cells prepared by the method.

[0090] Specifically, 110.sup.5210.sup.5 of human pluripotent stem cell-derived hematopoietic stem cells prepared according to the method of Example 1 were injected into a tail vein of irradiated immunodeficient NSG mice. The presence of human mitochondrial genes was confirmed in the peripheral blood of mice 12 weeks after injection, and the presence of human CD45-positive cells was confirmed in the peripheral blood of mice 22 weeks after injection (FIG. 7A).

[0091] PCR reactions were performed to detect human mitochondrial DNA in the peripheral blood of the mice, specifically, 2PCRBIO HS Taq Mix red was used as polymerase, and the annealing temperature was set to 58 C. and performed for 35 cycles. Afterwards, the PCR reaction product was loaded onto a 1% agarose gel containing EtBr at 100 v for 20 minutes to confirm the PCR band. The sequence of the primers used to detect human mitochondria is as follows.

TABLE-US-00001 Forward: (SEQIDNO:1) 5-CAACACTAAAGGACGAACCTGA-3 Reverse: (SEQIDNO:2) 5-TCGTAAGGGGTGGATTTTTC-3

[0092] As a result, human mitochondrial genes were detected in the peripheral blood of mice 12 weeks after injection (FIG. 7B), and 22 weeks after injection, it was confirmed that more than 25% of human CD45-positive cells were present in the peripheral blood of mice (FIG. 7C).

[0093] Based on the above results, it may be seen that the hematopoietic stem cells prepared by the hematopoietic stem cell differentiation protocol method of Example 1 may also be usefully used to construct a humanized animal model.

[0094] The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present disclosure may be easily modified into other specific forms without changing the technical concept or essential features of the present disclosure. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.