阅读说明：本技术 组合物在将造血祖细胞重编程为造血干细胞中的用途 (Use of compositions for reprogramming hematopoietic progenitor cells to hematopoietic stem cells ) 是由 肖雄 刘洪蛟 刘德芳 刘英全 严小娥 齐海龙 李迎霞 孙忠杰 陈立功 于 2019-11-05 设计创作，主要内容包括：本发明提出了组合物在将造血祖细胞重编程为造血干细胞中的用途,所述组合物包括：JNK-IN-8、丙戊酸和Y27632；以及任选的烟酰胺腺嘌呤二核苷酸。利用本发明的组合物可以将造血祖细胞重编程为造血干细胞,并且,获得的造血干细胞具有长期移植重建能力,能够稳定分化为T淋巴细胞、B淋巴细胞、髓系细胞、血红细胞等多个血细胞系,科学研究、临床研究和应用价值高。(The present invention proposes the use of a composition for reprogramming hematopoietic progenitor cells into hematopoietic stem cells, the composition comprising: JNK-IN-8, valproic acid and Y27632; and optionally nicotinamide adenine dinucleotide. The composition can reprogram hematopoietic progenitor cells into hematopoietic stem cells, and the obtained hematopoietic stem cells have long-term transplantation reconstruction capability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells and the like, and have high scientific research, clinical research and application values.)

1. A composition for reprogramming hematopoietic progenitor cells to hematopoietic stem cells, comprising:

JNK-IN-8, valproic acid and Y27632; and

optionally nicotinamide adenine dinucleotide.

2. A medium for reprogramming hematopoietic progenitor cells into 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 0.1 to 5 μ M, the concentration of nicotinamide adenine dinucleotide is 0.5 to 5 μ M, the concentration of valproic acid is 1 to 20nM, and the concentration of Y27632 is 1 to 20 μ M;

the basal medium is selected from the group consisting of a stemspan medium containing at least one of Flt3 ligand, thrombopoietin, stem cell growth factor, and low density lipoprotein;

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

4. A method of obtaining hematopoietic stem cells comprising: inhibiting at least one of the following metabolic pathways that produce blood progenitor cells: a JNK signal pathway, an HDAC signal pathway, and a ROCK signal pathway;

optionally, employing the composition of claim 1 so as to inhibit a JNK signaling pathway, an HDAC signaling pathway, and a ROCK signaling pathway;

optionally, the method comprises: culturing hematopoietic progenitor cells in the medium of claim 2 or 3.

5. A hematopoietic stem cell obtained by the method of claim 4.

6. A kit for reprogramming hematopoietic progenitor cells into hematopoietic stem cells, comprising: the composition of claim 1 or the culture medium of claim 2 or 3.

7. Use of a composition according to claim 1 for the preparation of an inhibitor for reprogramming hematopoietic progenitor cells to hematopoietic stem cells, inhibiting the metabolic pathways of at least one of the following:

the JNK signaling pathway, the HDAC signaling pathway, and the ROCK signaling pathway.

8. A pharmaceutical composition, comprising: the composition of claim 1, the culture medium of claim 2 or 3, or the hematopoietic stem cell of claim 5.

9. A method of screening for a drug comprising:

culturing the candidate drug with hematopoietic progenitor cells;

determining whether at least one of an intracellular JNK signaling pathway, an HDAC signaling pathway, and a ROCK signaling pathway is inhibited and hematopoietic stem cells are obtained before and after culturing;

an indication that the drug candidate is a drug of interest when at least one of the JNK signaling pathway, HDAC signaling pathway and ROCK signaling pathway is inhibited and hematopoietic stem cells are obtained after culturing,

the target drug is the composition of claim 1 or the culture medium of claim 2 or 3 or the pharmaceutical composition of claim 8.

Use of JNK-IN-8, valproic acid, Y27632, and optionally nicotinamide adenine dinucleotide for reprogramming hematopoietic progenitor cells to hematopoietic stem cells.

Technical Field

The invention relates to the field of biomedicine. In particular, the invention relates to the use of compositions for reprogramming hematopoietic progenitor cells into hematopoietic stem cells.

Background

Hematopoietic Stem Cells (HSCs) are the earliest stem cell type used in clinical therapy and have a strong self-renewal capacity and a high multilineage differentiation capacity. Since its successful application in the treatment of immunodeficiency in 1968, over the course of over 40 years of clinical improvement, the treatment of leukemia and other blood disorders by hematopoietic stem cell transplantation has been a relatively mature treatment (Barriga et al, 2012; Park et al, 2015). Hematopoietic stem cells are mainly derived from tissues such as human Bone Marrow (BM), mobilized Peripheral Blood (PB), and Umbilical Cord Blood (UCB), and HSCs derived from BM and PB are mainly used clinically. In recent years, as the amount of stored umbilical cord blood has increased, clinical applications of cord blood-derived HSCs have also become widespread. However, due to the limited number of HSCs extracted from one cord blood, the low probability of successful HLA matching of two, limits the clinical use of cord blood HSCs, and perhaps 50% of patients lose the opportunity to receive hematopoietic stem cell transplantation therapy (Boitano et al, 2010). Therefore, the expansion of hematopoietic stem cells in vitro has become a hot spot in this field.

In 2016, John Dick laboratory found that microRNA (microRNA-125 a) can amplify human and murine hematopoietic stem cells, and can reprogram hematopoietic progenitor cells into hematopoietic stem cells, thereby realizing the retrograde growth of blood cells for the first time and providing a new idea for obtaining hematopoietic stem cells in vitro (Wojtowicz et al, 2016). However, since this research work uses viral vectors, fragments are randomly inserted into the genome of cells, which poses a potential risk of canceration to the cells, thus greatly limiting the clinical application space. With the continuous development of chemical small molecule technology, the safety and reprogramming effect on cells are receiving wide attention. The research of expanding and reprogramming blood cells by adopting chemical micromolecules has safety guarantee, but until now, related research results are not reported.

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 a chemical micromolecule composition through a high-throughput screening platform, can reprogram hematopoietic progenitor cells into hematopoietic stem cells, and the obtained hematopoietic stem cells have long-term transplantation reconstruction capability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells and the like, and have high scientific research, clinical research and application values.

In one aspect of the invention, the invention features a composition for reprogramming hematopoietic progenitor cells into hematopoietic stem cells. According to an embodiment of the invention, the composition comprises: JNK-IN-8, valproic acid and Y27632; and optionally nicotinamide adenine dinucleotide.

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; nicotinamide Adenine Dinucleotide (NAD) can regulate basal metabolism and is a basal reaction substrate for cell growth; valproic acid (VPA) is an HDAC signaling pathway inhibitor; y27632 is ROCK1 inhibitor, can be used for resisting aging, and has effect in maintaining stem cell activity.

The inventors have employed high throughput screening to obtain small molecule compounds that can reprogram hematopoietic progenitor cells into hematopoietic stem cells, and have found that some chemical small molecules seem to be capable of reconstituting hematopoietic stem cells, but the obtained cells do not necessarily have hematopoietic stem cell functions, and have problems such as poor reconstitution capacity for transplantation, inability to differentiate into blood cell lineages such as T lymphocytes, B lymphocytes, myeloid cells, and red blood cells (T/B/M/E cells). The JNK-IN-8, the VPA and the Y27632 or the JNK-IN-8, the NAD, the VPA and the Y27632 have the effects of mutual matching and synergism, hematopoietic progenitor cells can be reprogrammed to be hematopoietic stem cells, the obtained hematopoietic stem cells have the long-term transplantation reconstruction capability, the reconstruction time is more than 6 months, the hematopoietic stem cells can be stably differentiated into a plurality of blood cell lines such as T/B/M/E and the like, and the scientific and clinical research and application values are high.

In another aspect of the invention, the invention features a medium for reprogramming hematopoietic progenitor cells into hematopoietic stem cells. According to an embodiment of the invention, the medium comprises: a basal medium; and the composition as described hereinbefore. Therefore, the culture medium according to the embodiment of the invention can effectively reprogram hematopoietic progenitor cells into hematopoietic stem cells, the obtained hematopoietic stem cells have long-term transplantation reconstruction capability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells and red blood cells, and has high scientific research, clinical research and application values.

According to an embodiment of the present invention, the above-mentioned medium for reprogramming hematopoietic progenitor cells into hematopoietic stem cells may further have the following additional technical features:

according to the embodiment of the invention, the concentration of the JNK-IN-8 is 0.1-5 mu M, the concentration of the nicotinamide adenine dinucleotide is 0.5-5 mu M, the concentration of the valproic acid is 1-20 nM, and the concentration of the Y27632 is 1-20 mu M. According to a specific embodiment of the invention, the concentrations of JNK-IN-8, nicotinamide adenine dinucleotide, valproic acid and Y27632 are 2. mu.M, 1. mu.M, 10nM, 10. mu.M or 3.5. mu.M, 0.8. mu.M, 15nM, 7. mu.M or 1.5. mu.M, 3. mu.M, 8nM, 15. mu.M, respectively. According to the specific embodiment of the invention, the concentration of the JNK-IN-8 is 1-3 mu M, the concentration of the nicotinamide adenine dinucleotide is 0.5-2 mu M, the concentration of the valproic acid is 6-15 nM, and the concentration of the Y27632 is 5-15 mu M. The inventors have made extensive experiments to obtain the above-mentioned preferable concentration, and thus obtained hematopoietic stem cells have a high long-term transplantation reconstitution ability and can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells, and the like.

According to an embodiment of the invention, the basal medium is selected from the group consisting of stemspan medium containing at least one of Flt3 ligand, Thrombopoietin (TPO), stem cell growth factor (SCF), and Low Density Lipoprotein (LDL). The present inventors have obtained the above-mentioned preferred composition through a large number of experiments, and thus have obtained hematopoietic stem cells having high long-term transplantation reconstitution ability and capable of stably differentiating into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells, and the like.

According to the embodiment of the invention, the concentration of the Flt3 ligand is 60-100 ng/mL, the concentration of thrombopoietin is 20-50 ng/mL, the concentration of stem cell factor is 60-100 ng/mL, and the concentration of low-density lipoprotein is 5-20 mug/mL. According to a specific embodiment of the invention, the concentrations of Flt3 ligand, thrombopoietin, stem cell factor and low density lipoprotein are 100ng/ml, 50ng/ml, 100ng/ml, 10. mu.g/ml or 80ng/ml, 30ng/ml, 80ng/ml, 15. mu.g/ml or 90ng/ml, 40ng/ml, 90ng/ml, 6. mu.g/ml, respectively. According to a specific embodiment of the invention, the concentration of the Flt3 ligand is 80-100 ng/mL, the concentration of thrombopoietin is 40-50 ng/mL, the concentration of stem cell factor is 80-100 ng/mL, and the concentration of low density lipoprotein is 5-15 mug/mL. The inventors have made extensive experiments to obtain the above-mentioned preferable concentration, and thus obtained hematopoietic stem cells have a high long-term transplantation reconstitution ability and can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells, and the like.

It should be noted that the type of the stemspan medium is not strictly limited in the present invention, and can be flexibly selected according to the actual situation. According to a particular embodiment of the invention, the StemSpan medium is selected from the StemSpan SFEM medium. Therefore, the effect is better.

According to embodiments of the invention, the invention provides JNK-IN-8, valproic acid and Y27632; and optionally nicotinamide adenine dinucleotide, in preparing a composition or culture medium for reprogramming hematopoietic progenitor cells into hematopoietic stem cells.

In yet another aspect of the invention, the invention features a method of obtaining hematopoietic stem cells. According to an embodiment of the invention, the method comprises: inhibiting at least one of the following metabolic pathways that produce blood progenitor cells: the JNK signaling pathway, the HDAC signaling pathway, and the ROCK signaling pathway. The inventors have found that inhibition of the JNK signaling pathway, HDAC signaling pathway and/or ROCK signaling pathway effectively reprograms hematopoietic progenitor cells into hematopoietic stem cells, and that the obtained hematopoietic stem cells have long-term transplantation reconstitution ability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells and red blood cells, and have a wide application prospect.

In the present invention, the substance or the mode of inhibition capable of inhibiting the JNK signaling pathway, HDAC signaling pathway and ROCK signaling pathway is not particularly limited as long as it can inhibit at least one of the three pathways to obtain hematopoietic stem cells, and specifically, it can be flexibly selected according to actual needs. According to an embodiment of the present invention, the above-described composition is employed so as to inhibit the JNK signaling pathway, HDAC signaling pathway, and ROCK signaling pathway. The inventors have conducted high-throughput screening analysis on a large number of small molecular substances capable of inhibiting a JNK signaling pathway, an HDAC signaling pathway and a ROCK signaling pathway, and found that hematopoietic stem cells can be obtained by using JNK-IN-8(JNK signaling pathway inhibitor), valproic acid (HDAC signaling pathway inhibitor) and Y27632(ROCK signaling pathway inhibitor) 3 factors or a combination of nicotinamide adenine dinucleotide and the obtained hematopoietic stem cells have long-term transplantation reconstitution capability.

According to an embodiment of the invention, the method comprises: hematopoietic progenitor cells are cultured in the medium described above.

In yet another aspect of the present invention, the present invention provides a hematopoietic stem cell. According to an embodiment of the present invention, the hematopoietic stem cells are obtained by the method for obtaining hematopoietic stem cells described above. Therefore, the hematopoietic stem cells according to the embodiments of the present invention have long-term transplantation reconstitution capability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells, and the like, and have a wide application prospect.

In yet another aspect of the invention, the invention features a kit for reprogramming hematopoietic progenitor cells into hematopoietic stem cells. According to an embodiment of the invention, the kit comprises: a composition as hereinbefore described for reprogramming hematopoietic progenitor cells to hematopoietic stem cells or a medium as hereinbefore described. Therefore, the kit according to the embodiment of the invention can effectively reprogram hematopoietic progenitor cells into hematopoietic stem cells, the obtained hematopoietic stem cells have long-term transplantation reconstruction capability, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells, red blood cells and the like, and has high 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 for reprogramming hematopoietic progenitor cells into hematopoietic stem cells, inhibiting the metabolic pathway of at least one of the following: the JNK signaling pathway, the HDAC signaling pathway, and the ROCK signaling pathway. As described above, JNK-IN-8, valproic acid and Y27632 or NAD can effectively inhibit a JNK signal pathway, an HDAC signal pathway and/or a ROCK signal pathway, and the hematopoietic progenitor cells are reprogrammed into hematopoietic stem cells, and the obtained hematopoietic stem cells have long-term transplantation reconstruction capability, the reconstruction time is more than 6 months, the hematopoietic stem cells can be stably differentiated into a plurality of blood cell lines such as T/B/M/E and the like, and the scientific and clinical research and application values are high.

In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the composition as described above, the culture medium as described above or the hematopoietic stem cells as described above. As described above, the composition according to the embodiment of the present invention can reprogram hematopoietic progenitor cells into hematopoietic stem cells, and directly or indirectly administer the composition or the culture medium as a pharmaceutical composition into a body (animal or cell) for the purpose of obtaining hematopoietic stem cells, or can apply the hematopoietic stem cells obtained by the method for obtaining hematopoietic stem cells into the 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: culturing the candidate drug with hematopoietic progenitor cells; determining whether at least one of an intracellular JNK signaling pathway, an HDAC signaling pathway, and a ROCK signaling pathway is inhibited and hematopoietic stem cells are obtained before and after culturing; when at least one of the JNK signaling pathway, HDAC signaling pathway and ROCK signaling pathway is inhibited and hematopoietic stem cells are obtained after culturing, is an indication that the drug candidate is a drug of interest, the drug of interest being the aforementioned composition or the aforementioned culture medium or the aforementioned pharmaceutical composition. As described above, the composition according to the embodiment of the present invention can inhibit at least one of the three metabolic pathways described above, thereby playing a role in reprogramming hematopoietic progenitor cells into hematopoietic stem cells. Meanwhile, the obtained hematopoietic stem cells have long-term transplantation reconstruction capability. 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 a further aspect of the invention, the invention proposes the use of JNK-IN-8, valproic acid, Y27632 and optionally nicotinamide adenine dinucleotide for reprogramming hematopoietic progenitor cells into hematopoietic stem cells. As described above, JNK-IN-8, valproic acid and Y27632 or JNK-IN-8, valproic acid, Y27632 and NAD can reprogram hematopoietic progenitor cells into hematopoietic stem cells, and the obtained hematopoietic stem cells have long-term transplantation reconstitution capacity, can be stably differentiated into a plurality of blood cell lines such as T lymphocytes, B lymphocytes, myeloid cells and red blood cells, and have wide application prospects.

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 above 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 of which:

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

FIG. 2 is a schematic diagram showing the results of flow cytometry analysis of hematopoietic stem cells cultured in a medium containing different small molecule compounds according to an embodiment of the present invention;

FIG. 3 shows a CD34 according to an embodiment of the invention⁺CD38⁺Schematic representation of in vitro phenotype identification analysis of reprogrammed cells at day 7 of cell culture;

FIG. 4 shows a schematic diagram of flow cytometric analysis of hematopoietic stem cells 20 weeks after reconstitution of hematopoietic stem cell transplantation according to an embodiment of the present invention;

FIG. 5 shows a schematic diagram of gene expression profiling analysis 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.