阅读说明：本技术 扩增造血干细胞的组合物、扩增方法、药物组合物和用途 (composition for expanding hematopoietic stem cells, expansion method, pharmaceutical composition and use ) 是由 孙忠杰 肖雄 刘英全 刘德芳 齐海龙 郭潇 王晓芳 陈立功 于 2019-11-05 设计创作，主要内容包括：本发明提出了扩增造血干细胞的组合物、扩增方法、药物组合物和用途,所述组合物由JNK-IN-8、雷帕霉素和Y27632组成。利用本发明的组合物可以高效扩增造血干细胞,有效维持造血干细胞的蛋白表型和基因表达谱,而且能够提高造血干细胞的体内重建功能和重建效率,具有重大的科学研究、临床研究和应用价值。(The invention provides a composition for amplifying hematopoietic stem cells, an amplification method, a pharmaceutical composition and application, wherein the composition consists of JNK-IN-8, rapamycin and Y27632. The composition can be used for efficiently amplifying the hematopoietic stem cells, effectively maintaining the protein phenotype and the gene expression profile of the hematopoietic stem cells, improving the in-vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and has important scientific research, clinical research and application values.)

1. A composition for expanding hematopoietic stem cells, comprising JNK-IN-8, rapamycin, and Y27632.

2. A medium for expanding hematopoietic stem cells, comprising:

a basal medium; and

The composition of claim 1.

3. the culture medium according to claim 2, wherein the concentration of JNK ~ IN ~ 8 is 1 ~ 5 μ M, the concentration of rapamycin is 1 ~ 20 nM, and the concentration of Y27632 is 1 ~ 20 μ M;

The basal medium is selected from StemBan SFEM medium containing Flt3 ligand, thrombopoietin, stem cell growth factor, and low density lipoprotein;

the concentration of the Flt3 ligand is 60 ~ 100ng/mL, the concentration of thrombopoietin is 20 ~ 50ng/mL, the concentration of stem cell factor is 60 ~ 100ng/mL, and the concentration of low ~ density lipoprotein is 5 ~ 20 mug/mL.

4. A method of expanding hematopoietic stem cells, comprising: the use of the composition of claim 1 to inhibit the following metabolic pathways of hematopoietic stem cells: JNK signal path, mTOR signal path and ROCK signal path.

5. the method of claim 4, wherein the method comprises: will CD34^＋culturing the cells in the medium of claim 2 or 3;

the CD34^＋the cells are derived from bone marrow, liver, spleen, peripheral blood or umbilical cord blood.

6. a kit for expanding hematopoietic stem cells, comprising: the composition of claim 1 or the culture medium of claim 2 or 3.

7. use of the composition of claim 1 for the preparation of an inhibitor for the expansion of hematopoietic stem cells, for the inhibition of the following metabolic pathways of hematopoietic stem cells:

JNK signal path, mTOR signal path and ROCK signal path.

8. A pharmaceutical composition, comprising: the composition of claim 1 or the culture medium of claim 2 or 3.

9. a method of screening for a drug comprising:

combining the candidate drug with CD34^＋Culturing the cells;

Determining whether a JNK signal pathway, an mTOR signal pathway and a ROCK signal pathway in the cells before and after culture are inhibited and/or whether the cells after culture are amplified and/or whether surface proteins representing the functions of hematopoietic stem cells in the cells before and after culture are consistent;

When the JNK signal pathway, the mTOR signal pathway and the ROCK signal pathway are inhibited after culture and/or after culture, cell amplification and/or surface proteins representing hematopoietic stem cell functions in cells before and after culture are consistent, the indication that the candidate drug is the target drug,

the target drug is the composition of claim 1 or the culture medium of claim 2 or 3.

Use of JNK-IN-8, rapamycin and Y27632 for expanding hematopoietic stem cells.

Technical Field

The invention relates to the field of biomedicine. In particular, the invention relates to the use of the composition for expanding hematopoietic stem cells.

Background

The hematopoietic stem cell transplantation technology is a common and effective treatment means for clinically treating various blood diseases and immune system diseases such as leukemia, lymphoma, aplastic anemia, thalassemia and the like. Hematopoietic stem cells are generally derived from three sources: bone marrow, peripheral blood and cord blood. Compared with bone marrow and peripheral blood hematopoietic stem cells, the umbilical cord blood hematopoietic stem cells are convenient to obtain, rich in sources, free of damage and side effects on donors, and therefore become an important source of hematopoietic stem cell transplantation donors.

At present, the bottleneck of cord blood hematopoietic stem cell transplantation technology is that the cell content is low, and the number of hematopoietic stem cells and progenitor cells contained in one cord blood is not enough to quickly restore the immune system of an adult patient, so that the fatality rate of opportunistic infection is increased. The current tentative strategy is transplantation of two umbilical cord blood, i.e. one patient receives transplantation of two umbilical cord blood after marrow removal, but this increases the difficulty of matching HLA of donors, so a method for amplifying umbilical cord blood hematopoietic stem cells is urgently needed to obtain sufficient hematopoietic stem cells for transplantation.

Therefore, how to effectively obtain enough hematopoietic stem cells in vitro becomes a scientific problem, and if the technical bottleneck can be broken through in the future, a new chapter of clinical application of the stem cells will be written.

Currently, the most important factor limiting the clinical use of hematopoietic stem cells is the insufficient number of HSCs. There are roughly three strategies to solve this problem: (1) directly differentiating by iPSC or ESC to obtain HSCs with functions; (2) obtaining HSCs by cell reprogramming; (3) existing HSCs are expanded in vitro. The two methods involve excessive technical difficulty and cause additional risks for clinical application, so that clinical research cannot be carried out at a later date. The expansion of hematopoietic stem cells in vitro is an important method for solving the problem.

The in vitro expansion of hematopoietic stem cells has many advantages, which are mainly summarized as the following three characteristics: (1) the homogeneity of the expanded cells is high, and the canceration risk is low; (2) the operation time for expanding HSC is relatively short, and more exogenous adverse factors are prevented from being introduced; (3) the starting cells are hematopoietic stem/progenitor cells, generally have no risk of causing tumor, and are safer in clinical application.

the studies of amplified HSCs can be broadly divided into two categories: (1) amplifying HSCs by using gene manipulation means such as introducing exogenous transcription factors or micro RNA; (2) HSCs are amplified by adopting chemical micromolecules. In recent years, with the continuous progress of chemical small molecule technology, small molecule compounds have been widely used in the field of stem cell research. The amplification of HSCs using small molecule drugs has a number of distinct advantages: low toxicity, easy elution and safer clinical use. However, there are also significant disadvantages, and the most worrying is that the target of action of small molecule compounds is not well defined, and there may be safety hazards, such as off-target effect of small molecules, so there still exists a lack of a safe and effective chemical small molecule combination scheme for expanding hematopoietic stem cells with well-defined target in clinic.

In summary, the technical scheme of using the combination of chemical small molecule compounds to expand hematopoietic stem cells in vitro is still under study.

Disclosure of Invention

the present invention aims to solve, at least to some extent, the technical problems of the prior art. Therefore, the invention finds some chemical small molecule compound combinations through a high-throughput screening platform, which not only can efficiently amplify the hematopoietic stem cells in vitro and effectively maintain the protein phenotype and the gene expression profile of the hematopoietic stem cells, but also can improve the in vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and has great scientific research, clinical research and application values.

In one aspect of the invention, the invention features a composition for expanding hematopoietic stem cells. According to an embodiment of the invention, the composition consists of JNK-IN-8, rapamycin, and Y27632.

JNK-IN-8 is a JNK signal pathway inhibitor, and the function of regulating and controlling a JNK signal pathway is realized by inhibiting c-Jun phosphorylation and gene transcription; rapamycin (Rapamycin) is an mTOR signaling pathway inhibitor useful for anti-aging and inhibiting tumor growth; y27632 is ROCK1 inhibitor, can be used for resisting aging, and has effect in maintaining stem cell activity.

The inventor adopts high-throughput screening to obtain a compound for efficiently amplifying hematopoietic stem cells, finds that 3 micromolecule compounds of JNK-IN-8, rapamycin and Y27632 have the effects of mutual matching and synergism, can efficiently amplify the hematopoietic stem cells, effectively maintains the protein phenotype and the gene expression profile of the hematopoietic stem cells, can improve the IN-vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and has great scientific research, clinical research and application values.

In another aspect of the invention, the invention features a medium for expanding hematopoietic stem cells. According to an embodiment of the invention, the medium comprises: a basal medium; and the composition for expanding hematopoietic stem cells as described above. Therefore, the culture medium provided by the embodiment of the invention can be used for efficiently amplifying the hematopoietic stem cells, effectively maintaining the protein phenotype and the gene expression profile of the hematopoietic stem cells, improving the in vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and has great scientific research, clinical research and application values.

according to an embodiment of the present invention, the above-mentioned culture medium may further have the following additional technical features:

according to the embodiment of the invention, the concentration of the JNK ~ IN ~ 8 is 1 ~ 5 mu M, the concentration of the rapamycin is 1 ~ 20 nM, and the concentration of the Y27632 is 1 ~ 20 mu M, according to the embodiment of the invention, the concentrations of the JNK ~ IN ~ 8, the rapamycin and the Y27632 are 2 mu M, 10nM, 10 mu M or 3.5 mu M, 15nM, 7 mu M or 1.5 mu M, 8nM and 15 mu M respectively, according to the embodiment of the invention, the concentration of the JNK ~ IN ~ 8 is 1 ~ 3 mu M, the concentration of the rapamycin is 6 ~ 15nM, and the concentration of the Y27632 is 5 ~ 15 mu M.

According to an embodiment of the invention, the basal medium is selected from the group consisting of StemBan SFEM medium containing Flt3 ligand, Thrombopoietin (TPO), stem cell growth factor (SCF), and Low Density Lipoprotein (LDL). Thus, the efficiency of amplification can be further improved by adding the above-mentioned factor to the StemBansfEM medium.

according to the embodiment of the invention, the concentration of the Flt3 ligand is 60- ~ ng/mL, the concentration of thrombopoietin is 20-50 ng/mL, the concentration of the stem cell factor is 60- ~ ng/mL, and the concentration of the low-density lipoprotein is 5-20 μ g/mL. according to the embodiment of the invention, the concentrations of Flt3 ligand, thrombopoietin, stem cell factor and low-density lipoprotein are ~ ng/mL, 50ng/mL, ~ ng/mL, 10 μ g/mL or 80ng/mL, 30ng/mL, 80ng/mL, 15 μ g/mL or 90ng/mL, 40ng/mL, 90ng/mL and 6 μ g/mL, respectively.

According to an embodiment of the invention, the invention proposes the use of JNK-IN-8, rapamycin and Y27632 IN the preparation of a composition or culture medium for the expansion of hematopoietic stem cells.

In yet another aspect of the present invention, the present invention provides a method for expanding hematopoietic stem cells. According to an embodiment of the invention, the method comprises: the following metabolic pathways of hematopoietic stem cells are inhibited using the compositions described above: JNK signal path, mTOR signal path and ROCK signal path. The inventor respectively carries out high-throughput screening analysis on a plurality of small molecular substances capable of inhibiting a JNK signal pathway, an mTOR signal pathway and a ROCK signal pathway, and finds that the combined use of the JNK-IN-8 (JNK signal pathway inhibitor), the rapamycin (mTOR signal pathway inhibitor) and the Y27632 (ROCK signal pathway inhibitor) 3 factor can efficiently amplify the hematopoietic stem cells, effectively maintain the protein phenotype and the gene expression spectrum of the hematopoietic stem cells, and can improve the IN-vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, the operation is simple and convenient, and the application prospect is wide.

According to an embodiment of the invention, the method comprises: will CD34^＋The cells are cultured in the medium for expanding hematopoietic stem cells described above. Therefore, the method can further improve the amplification efficiency, effectively maintain the protein phenotype and the gene expression profile of the hematopoietic stem cells, improve the in vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, is simple and convenient to operate, and has wide application prospect.

according to an embodiment of the present invention, the CD34^＋the cells are derived from bone marrow, liver, spleen, peripheral blood or umbilical cord blood.

in yet another aspect of the present invention, the present invention provides a kit for expanding hematopoietic stem cells. According to an embodiment of the invention, the kit comprises: the composition for expanding hematopoietic stem cells described above or the culture medium described above. Therefore, the kit provided by the embodiment of the invention can be used for efficiently amplifying the hematopoietic stem cells, effectively maintaining the protein phenotype and the gene expression profile of the hematopoietic stem cells, improving the in vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and has great scientific research, clinical research and application values.

In a further aspect of the invention, the invention proposes the use of a composition as hereinbefore described for the preparation of an inhibitor. According to an embodiment of the invention, the inhibitor is used for expanding hematopoietic stem cells, inhibiting the following metabolic pathways of hematopoietic stem cells: JNK signal path, mTOR signal path and ROCK signal path. As described above, JNK-IN-8, rapamycin and Y27632 can effectively inhibit a JNK signal pathway, an mTOR signal pathway and/or a ROCK signal pathway, thereby efficiently amplifying hematopoietic stem cells, effectively maintaining the protein phenotype and the gene expression profile of the hematopoietic stem cells, improving the IN vivo reconstruction function and the reconstruction efficiency of the hematopoietic stem cells, and having great scientific research, clinical research and application values.

in yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: a composition as hereinbefore described or a culture medium as hereinbefore described. As described above, the pharmaceutical composition according to the embodiment of the present invention can efficiently expand hematopoietic stem cells, and directly or indirectly administer the composition or the culture medium as a pharmaceutical composition into a body (animal or cell) to expand hematopoietic stem cells, or apply hematopoietic stem cells obtained by the method for expanding hematopoietic stem cells into a body, which has a good in vivo reconstitution function and reconstitution efficiency, can be widely used for the treatment of blood system diseases and autoimmune diseases, and has significant scientific research, clinical research and application values.

according to embodiments of the present invention, the pharmaceutical compositions of the present invention may be used in conjunction with conventional methods of treatment and/or therapy, or may be used separately from conventional methods of treatment and/or therapy. When the pharmaceutical compositions of the present invention are administered in combination therapy with other drugs, they may be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions of the present invention may also comprise a combination of a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and other therapeutic or prophylactic agents known in the art.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The mesenchymal stem cell of the present invention may be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration.

In yet another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: combining the candidate drug with CD34^＋Culturing the cells; determining whether intracellular JNK signaling pathway, mTOR signaling pathway and ROCK signaling pathway are inhibited before and after culturing and/or whether cells are culturedWhether the surface proteins representing the functions of hematopoietic stem cells in cells before and after amplification and/or culture are consistent; when the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway are inhibited after culturing and/or the cells are expanded after culturing and/or surface proteins representing hematopoietic stem cell functions in the cells before and after culturing are consistent, is an indication that the candidate drug is the target drug, which is the composition or the culture medium as described above. As described above, the composition according to the embodiment of the present invention can inhibit the above three metabolic pathways, thereby having the effect of expanding hematopoietic stem cells. Meanwhile, the protein phenotype and the gene expression profile of the hematopoietic stem cells can be effectively maintained after amplification. Therefore, by adopting the method for screening the drug according to the embodiment of the invention, the composition of the invention or the culture medium or the drug containing the composition can be effectively screened, and the method has great scientific research, clinical research and application values.

In the present invention, "identity" is used in the broad sense to mean "identity" in which the surface proteins representing the functions of hematopoietic stem cells in cells before and after culture are identical "and since there are many surface proteins representing the functions of hematopoietic stem cells, all the surface proteins are identical to each other by at least 80%, 85%, 90%, 95%, 99% or 100% before and after culture, and are considered to be" identity ".

IN yet another aspect of the invention, the invention features the use of JNK-IN-8, rapamycin, and Y27632 for expanding hematopoietic stem cells. As described above, JNK-IN-8, rapamycin and Y27632 can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of the hematopoietic stem cells, improve the IN vivo reconstruction function and reconstruction efficiency of the hematopoietic stem cells, and have significant scientific research, clinical research and application values.

additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken IN conjunction with the accompanying drawings, wherein 3F is an experimental group comprising three small molecules, JNK-IN-8, rapamycin and Y27632, and wherein for convenience of description:

FIG. 1 shows a schematic flow diagram of a process for expanding hematopoietic stem cells according to an embodiment of the present invention;

FIG. 2 shows DMSO group CD34 according to an embodiment of the invention⁺Flow cytometric analysis after day 7 of cell culture;

FIG. 3 shows a 3F group CD34 according to an embodiment of the invention⁺flow cytometric analysis after day 7 of cell culture;

FIG. 4 shows a CD34 according to an embodiment of the invention⁺flow cytometric analysis after day 7 of cell culture;

Fig. 5 shows an analysis of cells of hematopoietic stem cells according to an example of the invention after culturing in medium containing different small molecule compounds (control group DMSO conditions,. P <0.01,. P < 0.0001);

FIG. 6 is a schematic diagram showing the analysis of the expansion result of hematopoietic stem cells 20 weeks after reconstitution of hematopoietic stem cells according to an embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.