Use of a composition, media supplement or media in expanding hematopoietic stem cells or in the manufacture of a product of expanded hematopoietic stem cells

Abstract

This application provides the use of a composition, culture medium additive, or culture medium in expanding hematopoietic stem cells or preparing products containing expanded hematopoietic stem cells. A first aspect of this application discloses the use of a composition, culture medium additive, or culture medium in expanding hematopoietic stem cells or preparing products containing expanded hematopoietic stem cells. The composition includes quinsinostat and resveratrol; the culture medium additive includes the composition; the culture medium includes a basal medium and an additive, the additive including the composition. By screening specific HDAC inhibitors and combinations of various other small molecule compounds, the CD34 concentration of HSCs after in vitro expansion can be significantly increased at lower working concentrations. + CD34 + CD90 + and CD34 + CD90 + EPCR + The proportion of cells, that is, the proportion of hematopoietic stem cells and long-acting hematopoietic stem cells, can be increased.

Description

Technical Field

[0001] This application relates to the field of cell biology technology, and in particular to the use of compositions, culture medium additives or culture media in expanding hematopoietic stem cells or in the preparation of products containing expanded hematopoietic stem cells. Background Technology

[0002] Hematopoietic stem cells (HSCs) are self-renewing and multi-differentiating stem cells located in hematopoietic tissues. They can differentiate into various types of blood cells, including red blood cells, white blood cells, and platelets, thus maintaining the normal function of the blood and immune systems. Based on this principle, HSC transplantation has become an effective treatment for a variety of diseases, including hematological disorders, immune system diseases, and some metabolic genetic disorders. Currently, the main sources of HSCs include bone marrow, mobilized peripheral blood, and umbilical cord blood. Among these, peripheral blood HSC collection is simple and patient-friendly, but the proportion of HSCs in peripheral blood is very low. It requires the injection of granulocyte colony-stimulating factor (G-CSF) or plerixafor to mobilize hematopoietic stem cells from the bone marrow into the peripheral blood. Moreover, there is a risk of needing to repeat the procedure or even failure due to insufficient HSCs.

[0003] In clinical practice, HSC usually refers to CD34 in the blood. + Hematopoietic stem cells, including the most primitive hematopoietic stem cells (long-lived hematopoietic stem cells, LT-HSCs), short-lived hematopoietic stem cells, pluripotent progenitor cells, common myeloid progenitor cells, and common lymphoid progenitor cells, are various cell types with different self-renewal and differentiation potentials. However, only LT-HSCs can function long-term, while other stem cells gradually deplete. Based on the expression of surface proteins, LT-HSCs are generally defined as CD34... + CD38 - CD45RA - CD90 + The cells. But research has found that CD38 + Cells lose CD38 expression during in vitro culture, therefore CD38 is unsuitable for characterizing cultured HSCs. (Compared to CD34) + Compared to cells, CD34 + CD90 + The cell population contained a higher proportion of LT-HSCs, but the proportion was still low. Recent studies have found that EPCR can effectively characterize in vitro amplified LT-HSCs, and in vitro cultured LT-HSCs exhibit CD34... + CD90 + EPCR + Highly enriched within the group.

[0004] The success rate of hematopoietic stem cell (HSC) transplantation is closely related to the number of transplanted cells. When the number of transplanted HSCs is low, it takes longer to rebuild the blood and immune system, and the risk of infection increases. Besides collecting more cells, in vitro expansion of HSCs is also an effective way to address the clinical shortage of cell sources and quantities, and improve the success rate of hematopoietic stem cell transplantation. Related studies have explored various methods to achieve in vitro expansion of HSCs, with the most clinical progress being the addition of specific small molecule compounds to the in vitro culture of HSCs to promote their expansion and maintenance. For example, CD34 purified from mobilized peripheral blood... + Cells were cultured in vitro with 1 mM VPA, but the results showed that CD34 was present in the expanded cells. + The low cell proportion also failed to accurately characterize the proportion of long-acting hematopoietic stem cells after expansion. Furthermore, the high concentration may introduce more chemical residues into the cell product, posing certain safety risks. Therefore, it is necessary to provide a composition that can expand HSCs with a higher proportion of LT-HSCs at a lower working concentration. Summary of the Invention

[0005] The main objective of this invention is to propose the application of a composition, culture medium additive, or culture medium in the amplification of hematopoietic stem cells or the preparation of products containing amplified hematopoietic stem cells, which can amplify HSCs with a higher LT-HSC ratio at a lower working concentration.

[0006] To achieve the above objectives, a first aspect of this application provides the use of a composition, culture medium additive, or culture medium in expanding hematopoietic stem cells or in preparing products containing expanded hematopoietic stem cells, wherein the composition comprises quinsinostat and resveratrol; the culture medium additive comprises the composition, the culture medium comprises a basal culture medium and an additive, and the additive comprises the composition.

[0007] Quisinostat is a pan-histone deacetylase (HDAC) inhibitor with CAS number 875320-29-9 and its structural formula is as follows:

[0008] .

[0009] Resveratrol is a non-flavonoid polyphenol compound with various biological functions, including antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, immunomodulatory, regulation of glucose and lipid metabolism and inhibition of fat deposition, and protection of the cardiovascular system. Its CAS number is 501-36-0, and its structural formula is as follows:

[0010] .

[0011] In some implementations, the working concentration of quinsinositol is 0.1 to 5 nmol / L, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5 nmol / L.

[0012] The term "working concentration" refers to the concentration of the components used in the actual application of the composition. It may be stored and transported in a higher concentration solution, and then diluted to the appropriate working concentration before use. It is understood that other commonly used forms, such as dry powder, may also be used for storage and transport.

[0013] In some embodiments, the working concentration of the second component is 0.1~50 μmol / L, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μmol / L.

[0014] In some embodiments, the working concentration of resveratrol is 1 to 50 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μmol / L.

[0015] In some embodiments, the composition further includes at least one of tranylcyclopropane and pomalidomide.

[0016] Tranylcypromine (TCP) is an inhibitor of monoamine oxidase and lysine-specific demethylase 1 (LSD1), with CAS number 13492-01-8. Its structural formula is as follows:

[0017] .

[0018] Pomalidomide (POM) is a thalidomide analogue with significant effects in immune system regulation, anti-tumor activity, and inhibition of inflammatory responses. Its CAS number is 19171-19-8, and its structural formula is as follows:

[0019] .

[0020] In some embodiments, the working concentration of cyclopropane is 1 to 10 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μmol / L.

[0021] In some embodiments, the working concentration of pomalidomide is 1 to 10 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μmol / L.

[0022] In some embodiments, the composition includes quinsinol, resveratrol, and transphenylcyclopropionylamine. In some embodiments, the working concentration of quinsinol is 0.1–5 nmol / L, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5 nmol / L. In some embodiments, the working concentration of resveratrol is 1~50 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μmol / L. In some embodiments, the working concentration of transphenylcyclopropylamine is 1~10 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 μmol / L.

[0023] In some embodiments, the composition includes quinsinostamine, resveratrol, and pomalidomide. In some embodiments, the working concentration of quinsinostamine is 0.1–5 nmol / L, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5 nmol / L. In some embodiments, the working concentration of resveratrol is 1~50 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μmol / L. In some embodiments, the working concentration of pomalidomide is 1~10 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 μmol / L.

[0024] In some implementations, the basal culture medium also includes cytokines.

[0025] In some embodiments, the cytokines include at least one of stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), and interleukin-3 (IL-3).

[0026] In some embodiments, the working concentration of stem cell factors in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0027] In some embodiments, the working concentration of Fms-like tyrosine kinase 3 ligand in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0028] In some embodiments, the working concentration of thrombopoietin in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0029] In some implementations, the working concentration of interleukin-6 in the culture medium is 20 to 80 ng / mL, for example, 20, 30, 40, 50, 60, 70, or 80 ng / mL.

[0030] In some implementations, the working concentration of interleukin-3 in the culture medium is 5 to 20 ng / mL, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL.

[0031] In some implementations, the basal culture medium is a serum-free culture medium.

[0032] In some embodiments, the basal culture medium includes at least one of HIPP-T009 serum-free medium, SFEM serum-free medium, and SCGM serum-free medium.

[0033] In some embodiments, hematopoietic stem cells include at least one of umbilical cord blood hematopoietic stem cells, bone marrow hematopoietic stem cells, and peripheral blood hematopoietic stem cells.

[0034] In some implementations, peripheral blood hematopoietic stem cells are mobilized hematopoietic stem cells, including hematopoietic stem cells originally located in the peripheral blood as well as hematopoietic stem cells that have been mobilized from sources such as bone marrow into the peripheral blood circulation.

[0035] In some embodiments, the mobilization method includes at least one of mobilization by a mobilizing agent and mobilization by chemotherapy, such as mobilization by a mobilizing agent, mobilization by chemotherapy, or mobilization by a mobilizing agent combined with mobilization by chemotherapy. In some embodiments, the mobilizing agent includes cytokines (such as granulocyte colony-stimulating factor G-CSF), adhesion molecules, and / or antagonists of chemokines (such as CXC group chemokine receptor 4 antagonists such as praxavir). In some embodiments, the chemotherapeutic agents include cyclophosphamide, fludarabine, etc.

[0036] In some embodiments, the hematopoietic stem cells are animal-derived hematopoietic stem cells, such as those derived from mammals. Common mammals include rodents (e.g., mice, rats, hamsters, guinea pigs), lagomorphs (e.g., rabbits), perissodactyls (e.g., horses, donkeys), even-toed ungulates (e.g., sheep, goats, camels, cattle, pigs), primates (e.g., monkeys, gorillas, chimpanzees, humans), and carnivores (e.g., dogs, cats). In some embodiments, the hematopoietic stem cells are human-derived hematopoietic stem cells. In some embodiments, the hematopoietic stem cells are autologous or allogeneic hematopoietic stem cells. It is understood that allogeneic hematopoietic stem cells need to be pre-matched, for example, with HLA (human leukocyte antigen) matching.

[0037] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. +Cells. For example, CD34 in live cells of the amplification product. + The cell proportion is above 50%, for example, it can be 50%, 51%, 52%, 53%, 54%, or 55% or more. In some embodiments, the live cells of the amplified product contain CD34. + The proportion of cells is between 50% and 90%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0038] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. + CD90 + Cells, such as CD34 in living cells of the amplification product. + CD90 + The cell proportion is above 30%, for example, it can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% or more. In some embodiments, the live cells of the amplified product contain CD34. + CD90 + The proportion of cells is between 35% and 60%, for example, it can be 35%, 40%, 45%, 50%, 55%, or 60%.

[0039] In some implementations, the CD34 of the hematopoietic stem cell amplification products... + CD34 in cells + CD90 + The cell percentage is above 5%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In some embodiments, the CD34 of the amplified product... + CD34 in cells + The proportion of cells is between 50% and 88%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 88%.

[0040] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. + CD90 + EPCR + Cells. For example, CD34 in live cells of the amplification product. + CD90 + EPCR + The cell percentage is above 1%, for example, it could be 1%, 2%, 3%, 4%, or 5% or higher. In some embodiments, the live cells of the amplified product contain CD34. + CD90 + EPCR +The proportion of cells is between 2% and 28%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%.

[0041] In some implementations, the CD34 of the hematopoietic stem cell amplification products... + CD90 + CD34 in cells + CD90 + EPCR + The cell proportion is above 5%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10% or more. In some embodiments, the CD34 of the amplified product... + CD90 + CD34 in cells + CD90 + EPCR + The proportion of cells ranges from 5% to 48%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%.

[0042] In some implementations, the application includes enriching CD34 from the expansion products of hematopoietic stem cells. + cell.

[0043] In some implementations, the application includes enriching CD34 from the expansion products of hematopoietic stem cells. + CD90 + cell.

[0044] In some implementations, the application includes enriching CD34 from the expansion products of hematopoietic stem cells. + CD90 + EPCR + cell.

[0045] In some implementations, the application is for purposes other than disease diagnosis or treatment.

[0046] Another aspect of this application provides a method for expanding hematopoietic stem cells, the method comprising the step of culturing the hematopoietic stem cells to be expanded in a culture medium;

[0047] The culture medium includes the aforementioned composition, the aforementioned culture medium additive, or the aforementioned culture medium.

[0048] In some embodiments, hematopoietic stem cells include at least one of umbilical cord blood hematopoietic stem cells, bone marrow hematopoietic stem cells, and peripheral blood hematopoietic stem cells.

[0049] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. + Cells. For example, CD34 in live cells of the amplification product. + The cell proportion is above 50%, for example, it can be 50%, 51%, 52%, 53%, 54%, or 55% or more. In some embodiments, the live cells of the amplified product contain CD34. + The proportion of cells is between 50% and 90%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0050] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. + CD90 + Cells, such as CD34 in living cells of the amplification product. + CD90 + The cell proportion is above 30%, for example, it can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% or more. In some embodiments, the live cells of the amplified product contain CD34. + CD90 + The proportion of cells is between 35% and 60%, for example, it can be 35%, 40%, 45%, 50%, 55%, or 60%.

[0051] In some implementations, the CD34 of the hematopoietic stem cell amplification products... + CD34 in cells + CD90 + The cell percentage is above 5%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In some embodiments, the CD34 of the amplified product... + CD34 in cells + The proportion of cells is between 50% and 88%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 88%.

[0052] In some implementations, the expansion products of hematopoietic stem cells are enriched with CD34. + CD90 + EPCR + Cells. For example, CD34 in live cells of the amplification product. + CD90 + EPCR+ The cell percentage is above 1%, for example, it could be 1%, 2%, 3%, 4%, or 5% or higher. In some embodiments, the live cells of the amplified product contain CD34. + CD90 + EPCR + The proportion of cells is between 2% and 28%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%.

[0053] In some implementations, the CD34 of the hematopoietic stem cell amplification products... + CD90 + CD34 in cells + CD90 + EPCR + The cell proportion is above 5%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10% or more. In some embodiments, the CD34 of the amplified product... + CD90 + CD34 in cells + CD90 + EPCR + The proportion of cells ranges from 5% to 48%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%.

[0054] In some embodiments, the method includes enriching CD34 from the expansion products of hematopoietic stem cells. + cell.

[0055] In some embodiments, the method includes enriching CD34 from the expansion products of hematopoietic stem cells. + CD90 + cell.

[0056] In some embodiments, the method includes enriching CD34 from the expansion products of hematopoietic stem cells. + CD90 + EPCR + cell.

[0057] In some embodiments, the hematopoietic stem cells to be expanded are cultured in the culture medium for 5 to 10 days, for example, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the medium is partially changed every 2 to 4 days during the culture period, for example, every 2, 3, or 4 days.

[0058] In some embodiments, the culture medium includes a basal culture medium and an additive, the additive including a composition.

[0059] In some embodiments, the composition includes quinsinostat and resveratrol.

[0060] In some implementations, the working concentration of quinsinositol is 0.1 to 5 nmol / L, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5 nmol / L.

[0061] In some embodiments, the working concentration of resveratrol is 0.1~50 μmol / L, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μmol / L.

[0062] In some embodiments, the composition further includes at least one of tranylcyclopropane and pomalidomide.

[0063] In some embodiments, the working concentration of at least one of cyclopropanediolamine and pomalidomide is 1 to 10 μmol / L, for example, it can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μmol / L.

[0064] In some embodiments, the composition includes quinsinostat, resveratrol, and transphenylcyclopropane.

[0065] In some embodiments, the composition includes quinsinostat, resveratrol, and pomalidomide.

[0066] In some implementations, the basal culture medium also includes cytokines.

[0067] In some embodiments, the cytokines include at least one of stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), interleukin-6 (IL-6), and interleukin-3 (IL-3).

[0068] In some embodiments, the working concentration of stem cell factors in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0069] In some embodiments, the working concentration of Fms-like tyrosine kinase 3 ligand in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0070] In some embodiments, the working concentration of thrombopoietin in the culture medium is 30 to 150 ng / mL, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL.

[0071] In some implementations, the working concentration of interleukin-6 in the culture medium is 20 to 80 ng / mL, for example, 20, 30, 40, 50, 60, 70, or 80 ng / mL.

[0072] In some implementations, the working concentration of interleukin-3 in the culture medium is 5 to 20 ng / mL, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL.

[0073] In some implementations, the basal culture medium is a serum-free culture medium.

[0074] In some embodiments, the basal culture medium includes at least one of HIPP-T009 serum-free medium, SFEM serum-free medium, and SCGM serum-free medium.

[0075] In some implementations, the method is for purposes other than disease diagnosis or treatment.

[0076] The beneficial effects of this invention are:

[0077] By screening specific HDAC inhibitors and combinations of various other small molecule compounds, the CD34 concentration of HSCs after in vitro amplification can be significantly improved at lower working concentrations. + CD34 + CD90 + and CD34 + CD90 + EPCR + The proportion of cells, that is, the proportion of hematopoietic stem cells and long-acting hematopoietic stem cells, can be increased. Attached Figure Description

[0078] Figure 1 This is Example 1 of the present application, which describes the flow cytometry analysis of cell surface marker expression. In this example, P1 represents live cells, and P2 represents CD34 in live cells. + The proportion of cells, P3 is CD34 + CD90 in cells + The ratio of cells, P2 and P3 multiplied together, gives the CD34 content in living cells. + CD90 + The proportion of cells, P4 being CD34 + CD90 + CD34 in cells + CD90 + ECPR + The ratio of cells, P2, P3, and P4, multiplied together gives the CD34 content in living cells. + CD90 + EPCR + The proportion of cells.

[0079] Figure 2 This refers to the CD34 derived from mobilized peripheral blood in Embodiment 1 of this application. + Flow cytometry results of in vitro cell expansion culture. Where A represents CD34 in live cells. + The proportion of cells, where B represents CD34 in living cells. + CD90 + The proportion of cells, where C represents CD34 in living cells. + CD90 + EPCR + The proportion of cells, where D is the fold increase in total nucleated cells (TNCs) after treatment with several different HDAC inhibitors.

[0080] Figure 3 The combination of two different small molecule compounds in Example 2 of this application is effective against CD34 from mobilized peripheral blood. + Flow cytometry results of in vitro cell expansion culture. In this figure, A, B, and C represent CD34, etc. + Cells, CD34 + CD90 + Cells and CD34 + CD90 + EPCR + The proportion of cells.

[0081] Figure 4 The combination of three different small molecule compounds in Example 3 of this application is effective against CD34 from mobilized peripheral blood. +Flow cytometry results of in vitro cell expansion culture. In this figure, A, B, and C represent CD34, etc. + Cells, CD34 + CD90 + Cells and CD34 + CD90 + EPCR + The proportion of cells. Detailed Implementation

[0082] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing embodiments of this application only and is not intended to limit the scope of this application.

[0083] As those skilled in the art will recognize, with the evolution of technology and the emergence of new application scenarios, the technical solutions provided in the embodiments of this application are also applicable to similar technical problems.

[0084] The raw materials and their manufacturers' part numbers involved in the following examples are shown in Table 1:

[0085] Table 1. List of Raw Materials and Reagents

[0086]

[0087] Example 1: Screening of small molecule compounds

[0088] 1. Experimental Procedure

[0089] (1) Preparation of small molecule compound solutions: Prepare high-concentration small molecule compound storage solutions according to the solubility and corresponding solvents listed in the instructions for small molecule compounds. Based on the final concentration of each small molecule, dilute the storage solutions with the solvent to obtain a stock solution of the corresponding concentration. See Table 1 for the various small molecule compounds, numbers 1 to 19.

[0090] (2) Preparation of basic culture medium: 100 ng / mL SCF, 100 ng / mL LFLT3L, 100 ng / mL TPO, 50 ng / mL IL-6, 10 ng / mL IL-3 and 1% penicillin / streptomycin solution were added to HIPP-T009 culture medium respectively.

[0091] (3) Prepare a culture medium containing small molecule compounds: Add the stock solution of small molecule compounds from step (1) to the basic culture medium in proportion and mix well.

[0092] (4) Add 980 μL of the culture medium prepared in step (2) to a 24-well plate (NEST, 702012) that has not undergone tissue culture treatment. Remove frozen human mobilized peripheral blood HSCs (sorted CD34 cells) from liquid nitrogen. + Cells were rapidly thawed in a 37°C water bath. The cryopreservation solution containing cells was transferred to a 15 mL centrifuge tube, five volumes of DPBS were added, and the tube was centrifuged at 400 g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in the basal culture medium described earlier. Cells were counted, and the cell density was adjusted to 2 × 10⁶ cells / mL using basal culture medium. 6 Add 20 μL of cell suspension to each well, mix well, and incubate in a CO2 incubator (Thermo, model 3111). Perform a half-change of medium every 3 days.

[0093] 2. Flow cytometry detection

[0094] (1) Prepare flow cytometry buffer: Add 2% FBS and 1 mM EDTA (pH=8.0) to DPBS.

[0095] (2) After culturing the cells in the incubator for 9 days, the cells were counted. Based on the counting results, 1×10⁶ cells were taken from each sample. 5 One cell was transferred to a 1.5 mL centrifuge tube, and an equal number of cells were taken out at the same time to serve as a negative control (NC group: gate, to determine the range of positive cells) and a single staining group (adjusting the compensation between different fluorescence channels).

[0096] (3) Centrifuge the cells at 400 g for 5 min and discard the supernatant; add 1 mL of pre-cooled flow cytometry buffer to resuspend the cells, centrifuge at 400 g for 5 min and discard the supernatant; resuspend the cells with 20 μL of flow cytometry buffer, add 1 μL of Fc receptor blocking solution (Biolegend, 422302) and mix well, incubate at room temperature for 10 min, add the corresponding fluorescent antibody according to Table 2, mix well, and incubate at 4 ℃ in the dark for 30 min.

[0097] (4) Add 1 mL of pre-cooled flow cytometry buffer to each sample, centrifuge at 400 g for 5 min, discard the supernatant, repeat twice, resuspend the cell pellet with 100 μL of flow cytometry buffer, transfer the cells to a flow cytometer with a filter, and detect the sample by flow cytometer.

[0098] The detection process is illustrated below: First, the NC samples are tested. The viable cell population (P1) is circled in the FSC / SSC plot. Within P1 cells, the positive groups for the corresponding channels are circled. Single-stain tubes for FITC, PE, and APC are tested, and compensation is adjusted to eliminate interference between FITC, PE, and APC channels. Samples from each treatment group are then tested. CD34 is circled in the PE histogram of P1. + In group (P2), circle CD34 in the FITC histogram of P2. + CD90 + Circle CD34 in the APC histogram of group (P3). + CD90 + EPCR + Group (P4) is defined, and the proportions of P2, P3, and P4 in P1 are calculated.

[0099] The flow cytometry staining protocols are shown in Table 2:

[0100] Table 2. Flow cytometry staining protocols

[0101]

[0102] 3. Experimental Results

[0103] The small molecule compounds in Table 1 were subjected to treatment tests.

[0104] In preliminary tests, some small molecule compounds showed significant inhibitory effects on cell proliferation at the concentrations recommended in the literature (e.g., 1 mM VPA). Therefore, the test concentrations were reduced during the testing process. The test concentrations of different small molecule compounds are shown in Table 3.

[0105] Table 3. Final concentrations of different small molecule compounds in the culture medium

[0106]

[0107] Results of flow cytometry (TCP) Figure 1 As shown, P1 represents a live cell, and P2 represents CD34 in a live cell. + The proportion of cells, P3 is CD34 + CD90 in cells + The ratio of cells, multiplied by the ratio, gives the CD34 content in living cells. + CD90 + The proportion of cells, P4 being CD34 + CD90 + In cells, CD34 + CD90 + ECPR + The ratio of cells, multiplied by the product of these three factors, gives the CD34 content in living cells. +CD90 + EPCR + Cell ratio. For example, according to... Figure 1 CD34 in living cells + CD90 + The proportion of cells was 55.87% × 15.02%, approximately 8.39%, and the live cells contained CD34. + CD90 + EPCR + The proportion of cells is 55.87% × 15.02% × 4.14%, which is approximately 0.35%.

[0108] The screening results of small molecule compounds are as follows: Figure 2 It can be seen that various small molecule compounds can significantly increase CD34 levels after culture. + The proportion of cells, among which three HDAC inhibitors (VPA, SAHA, and Quisinostat) significantly increased CD34 levels after culture. + CD90 + and CD34 + CD90 + EPCR + The proportion of cells. For example... Figure 2 As shown in Figure D, the addition of VPA and SAHA resulted in a more significant decrease in the total number of nucleated cells than that of Quisinostat, indicating that VPA and SAHA have a more significant inhibitory effect on cell proliferation compared to Quisinostat. Figure 2 The calculation of results A, B, and C, and the CD34 amplified by Quisinostat + CD90 + EPCR + The number of cells was 1.8 times that of VPA and 2.8 times that of SAHA, respectively, so Quisinostat was chosen for subsequent screening of small molecule compound combinations.

[0109] Example 2: Screening of two-component combinations

[0110] Other small molecule compounds listed in Table 3 of Example 1 were combined with Quisinostat, and CD34 was detected according to the concentrations in Table 3 and the experimental procedures and flow cytometry methods described in Example 1. + CD34 + CD90 + and CD34 + CD90 + EPCR + The number of cells.

[0111] The results are as follows Figure 3As shown, compared with Quisinostat alone, Quisinostat+CA77.1, Quisinostat+FSK, Quisinostat+UNC0638, Quisinostat+SR-1, Quisinostat+Resveratrol, and Quisinostat+Repsox can all further improve CD34. + and CD34 + CD90 + The proportion of cells was significantly increased, with the combinations of Quisinostat+SR-1 and Quisinostat+Resveratrol showing the most significant effects. Furthermore, compared to the control group without small molecules, the combinations of Quisinostat+CA77.1, Quisinostat+FSK, Quisinostat+UNC0638, Quisinostat+SR-1, Quisinostat+Resveratrol, and Quisinostat+Repsox resulted in increased CD34 cell count. + CD90 + EPCR + The proportion of cells increased more than 10-fold, with the Quisinostat + Resveratrol combination increasing CD34 + CD90 + EPCR + The proportion of cells increased 50 times.

[0112] Example 3

[0113] Several other small molecule compounds from Example 2 were combined with Quisinostat + Resveratrol, and CD34 was detected according to the concentrations in Table 3 of Example 1, using the experimental procedure and flow cytometry method described in Example 1. + CD34 + CD90 + and CD34 + CD90 + EPCR + The number of cells.

[0114] The results are as follows Figure 4 As shown in the figure, the combinations Quisinostat+Resveratrol+TCP and Quisinostat+Resveratrol+POM maintain a high CD34 level. + While increasing cell ratio, it further improved CD34 + CD90 + and CD34 + CD90+ EPCR + Cell proportion. Compared with the Quisinostat+Resveratrol combination, the Quisinostat+Resveratrol+TCP and Quisinostat+Resveratrol+POM combinations resulted in CD34 + CD90 + EPCR + The proportions of cells increased by 5 times and 4 times, respectively.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a composition, culture medium additive, or culture medium in expanding peripheral blood hematopoietic stem cells or preparing products for the purpose of expanding peripheral blood hematopoietic stem cells for non-disease diagnosis or treatment purposes, characterized in that, The composition comprises quinsinostat and resveratrol; the culture medium additive comprises the composition; the culture medium comprises a basal medium and an additive, the additive comprising the composition, wherein the peripheral blood hematopoietic stem cell amplification product has a higher proportion of long-acting hematopoietic stem cell CD34. + CD90 + EPCR + Cells; among which, the working concentration of quinsinostat is 0.5~5 nmol / L, and the working concentration of resveratrol is 5~50 μmol / L.

2. The application according to claim 1, characterized in that, The composition further includes at least one of tranylcyclopropane and pomalidomide; wherein the working concentration of tranylcyclopropane is 1~10 μmol / L and the working concentration of pomalidomide is 1~10 μmol / L.

3. The application according to claim 1, characterized in that, The composition comprises quinsinusstat, resveratrol, and tranylcyclopropane; wherein the working concentration of quinsinusstat is 0.5-5 nmol / L, the working concentration of resveratrol is 5-50 μmol / L, and the working concentration of tranylcyclopropane is 1-10 μmol / L.

4. The application according to claim 1, characterized in that, The composition comprises quinsinusstat, resveratrol, and pomalidomide; wherein the working concentration of quinsinusstat is 0.5-5 nmol / L, the working concentration of resveratrol is 5-50 μmol / L, and the working concentration of pomalidomide is 1-10 μmol / L.

5. The application according to claim 1, characterized in that, The basal culture medium also includes cytokines; the cytokines include at least one of stem cell factor, Fms-like tyrosine kinase 3 ligand, thrombopoietin, interleukin-6, and interleukin-3.

6. The application according to claim 1, characterized in that, The expansion of peripheral blood hematopoietic stem cells includes enriching CD34 from the expansion products of the peripheral blood hematopoietic stem cells. + CD90 + EPCR + cell.

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