Use of iwr-1-endo and a-83-01 in promoting expansion and enhanced functionality of human circulating hematopoietic stem / progenitor cells

Abstract

The application provides applications of IWR-1-endo and A-83-01 in promoting expansion and enhancing functions of human circulating hematopoietic stem / progenitor cells, relates to the technical field of stem cell biology and regenerative medicine, and IWR-1-endo and / or A-83-01 can promote expansion of cHSPCs; compared with a single reagent, the combination of the two has a synergistic promoting effect, significantly promotes expansion of cHSPCs, and maintains cell stemness and regenerative capacity, thereby providing a new idea for solving the technical problems in the prior art that human circulating hematopoietic stem / progenitor cells are difficult to be rapidly expanded in ex vivo culture while maintaining cell stemness and regenerative potential.

Description

Technical Field

[0001] This invention relates to the fields of stem cell biology and regenerative medicine, and in particular to the application of small molecule compounds IWR-1-endo and A-83-01 in promoting the expansion and enhancing the function of human circulating hematopoietic stem / progenitor cells. Background Technology

[0002] Human circulating hematopoietic stem / progenitor cells (cHSPCs), as an easily accessible source of autologous stem cells, hold great potential in cell therapy. However, their extremely low abundance and rapid differentiation and loss of function under conventional in vitro culture conditions severely limit their clinical application. Existing expansion strategies mostly focus on adding cytokines or regulating a single pathway, but they cannot effectively maintain the self-renewal and transplantation capacity of the most primitive long-term hematopoietic stem cells (LT-HSCs).

[0003] Human circulating hematopoietic stem / progenitor cells (cHSPCs), as an easily accessible source of autologous stem cells, have shown great clinical application potential in the field of cell therapy. Compared with hematopoietic stem cells derived from bone marrow or umbilical cord blood, cHSPCs can be obtained from peripheral blood through a simple leukocyte ablation procedure, avoiding invasive tissue puncture and significantly reducing donor risk and operational difficulty, providing a safer and more accessible cell source for patients requiring autologous stem cell transplantation. Furthermore, the acquisition process of cHSPCs does not involve ethical controversies and can be repeated, laying the foundation for the large-scale application of cell therapy. However, the clinical application of cHSPCs still faces serious challenges: their abundance in the circulating system is extremely low, and once cultured in vitro, they rapidly lose their self-renewal capacity and differentiate into various blood cell lineages, leading to the depletion of the most therapeutically valuable long-term hematopoietic stem cells (LT-HSCs) within days, making it difficult to rebuild durable and stable multi-lineage hematopoietic function after transplantation.

[0004] Current strategies for expanding cHSPCs primarily rely on adding combinations of exogenous cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand (Flt-3L), to mimic the hematopoietic support signals of the bone marrow microenvironment. However, while these strategies can promote cell proliferation, they struggle to maintain the original phenotype and function of LT-HSCs. Studies have shown that cytokine-driven expansion is often accompanied by the directed differentiation of hematopoietic stem cells into progenitor cells, resulting in a predominance of short-term progenitor cells in the cell population and a significant decline in long-term regeneration capacity. More critically, single-cytokine regulation cannot mimic the complex signaling network integration within the in vivo hematopoietic stem cell microenvironment, neglecting the decisive influence of the synergistic effects of multiple signaling pathways in the microenvironment on rapid expansion, maintenance of cell stemness, and regenerative potential.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of small molecule compounds IWR-1-endo and / or A-83-01 in the expansion and enhancement of human circulating hematopoietic stem / progenitor cells, so as to solve the technical problem in the prior art that human circulating hematopoietic stem / progenitor cells are difficult to rapidly expand in vitro culture while maintaining cell stemness and regenerative potential.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of small molecule compounds in A1 to A4: A1. To prepare products for the expansion of human circulating hematopoietic stem / progenitor cells; A2. To prepare products for improving the regenerative capacity of human circulating hematopoietic stem / progenitor cells; A3. Prepare products for maintaining the stemness of human circulating hematopoietic stem / progenitor cells; A4. Prepare products for hematopoietic reconstitution; The small molecule compounds include IWR-1-endo and / or A-83-01.

[0008] Furthermore, the working concentration of the IWR-1-endo is 0.1~10 μM.

[0009] Furthermore, the working concentration of A-83-01 is 0.1~5μM.

[0010] Furthermore, the hematopoietic reconstitution includes at least one of B1 to B3: B1. Treatment of hematologic malignancies; B2. Treatment of hereditary blood diseases; B3. Restore hematopoietic function after chemotherapy and / or radiotherapy; Preferably, the product for hematopoietic reconstitution comprises a population of human circulating hematopoietic stem / progenitor cells obtained by amplification of human circulating hematopoietic stem / progenitor cells.

[0011] In a second aspect, the present invention provides a composition for in vitro expansion of human circulating hematopoietic stem / progenitor cells, comprising IWR-1-endo and / or A-83-01.

[0012] Furthermore, the final concentration of the IWR-1-endo is 0.1~10 μM; Preferably, the final concentration of A-83-01 is 0.1~5μM.

[0013] Thirdly, the present invention provides a culture medium for in vitro expansion of human circulating hematopoietic stem / progenitor cells, comprising the above-described composition.

[0014] Furthermore, the culture medium also includes basal culture medium and hematopoietic support factors; Preferably, the hematopoietic support factors include SCF, TPO, FLT3L, IL-3, and IL-6; Preferably, the working concentrations of SCF, TPO, and FLT3L are each independently 50~100 ng / mL; Preferably, the working concentrations of IL-3 and IL-6 are each independently 5~20 ng / mL.

[0015] Fourthly, the present invention provides a method for in vitro expansion of human circulating hematopoietic stem / progenitor cells for non-disease diagnosis and treatment purposes, comprising culturing peripheral blood mononuclear cells containing cHSPCs using the above-described culture medium.

[0016] Preferably, the culture time is 5 to 10 days; Preferably, the culture medium is replaced every 2-3 days during the culture period.

[0017] Fifthly, the present invention provides a human circulating hematopoietic stem / progenitor cell population, which is obtained by the above-described method.

[0018] The present invention provides the application of the small molecule compounds IWR-1-endo and / or A-83-01 in the expansion and enhancement of human circulating hematopoietic stem / progenitor cells. Both the small molecule compounds IWR-1-endo and the A-83-01 inhibitor can promote the expansion of cHSPCs. Compared with single reagents, the combination of small molecule compounds IWR-1-endo and A-83-01 has a synergistic promoting effect, significantly promoting the expansion of cHSPCs, maintaining cell stemness and regeneration capacity, and providing a new approach to solving the technical problem of the difficulty in rapidly expanding human circulating hematopoietic stem / progenitor cells in in vitro culture while maintaining cell stemness and regeneration potential. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the NISS experiment for conventional culture-induced inflammatory signaling network of cHSPC amplification and function provided in Example 1 of the present invention; Figure 2The figure shows the experimental results of the effect of the niche-induced inflammatory signaling network on cHSPC amplification and functional inhibition provided in Example 1 of this invention. It illustrates the effect of cHSPC culture alone and co-culture with PBMC helper cells for 7 days on LIN. - CD45RA - CD34 + CD38 low / - A quantitative statistical chart of cHSPC proportions, where A represents cHSPC culture alone and B represents cHSPC co-culture with PBMC helper cells. The data are expressed as mean ± SEM; n = 3 donors. Figure 3 CD34 derived from the cHSPC single-culture and co-culture systems provided in Example 1 of this invention + The results of differential gene analysis of cells show the results of differential gene enrichment in individual culture systems. Among them, A is the top KEGG pathway, B is the upregulated inflammatory factor interaction network, and C is the comparison of the expression levels of some factors occupying the main hub positions in the interaction network, n=3. Figure 4 To demonstrate the functional impact of the niche-induced inflammatory signaling network NISS provided in Example 1 of this invention on long-term implantation capability, n=12-13 mice; Figure 5 This is a schematic diagram of the screening process for small molecule compounds to screen potential NISS mitigators provided in Embodiment 2 of the present invention; Figure 6 This is the statistical result of the number of nucleated cells after treating cHSPCs with the small molecule compound to be screened, as provided in Example 2 of the present invention; Figure 7 The transcriptome sequencing analysis results after treating cHSPCs with molecular compounds provided in Example 2 of this invention; Figure 8 The expression levels of stemness, inflammation, and hematopoietic differentiation markers in cHSPCs after IWR-1-endo treatment provided in Example 2 of this invention are shown, where A is CD34, B is IFNG, MPO, MS4A1, SP11, IRF8, CD79A, CD79B, CD8A, C is CD83, CD79B, CD2, CD3D, D is CD38, CD86, CD4, CD3G, CD40, CD48, CD36, CD8A, CD5, and E is CD69, CD3E, CD79A, CD7, CD19. Figure 9 Validation of the ability of IWR-1-endo-amplified cHSPCs to reconstitute in vitro and in vivo. A is a schematic diagram of the experiment investigating the effects of IWR-1-endo on cHSPC amplification and function. B shows the effects of different Wnt pathway inhibitors on LIN. -CD45RA - CD34 + CD38 low / - The effect of fold changes in cHSPCs subpopulations, with n=3 donors for each condition; Figure 10 Validation of the ability of IWR-1-endo-amplified cHSPCs to reconstitute in vitro and in vivo (Part II). A shows the effect of different doses of IWR-1-endo on TNC amplification (n=3), and B shows the effect of different doses of IWR-1-endo on the amplification of 300 sorted CD34 cells after 7 days of culture. + Colony-forming unit (CFU) experiments were conducted using cells, n=3. C represents statistical analysis of the colony-forming data; GM represents granulocyte-macrophage CFU; CFU-E represents erythroid CFU; GEMM represents granulocyte-erythroid-macrophage-megakaryocyte CFU; and D represents human CD45 in peripheral blood. + Changes in cell chimerism over 4 months, n = 10–27 mice, E represents multi-lineage reconstitution 4 months post-transplantation; Figure 11 This is data from Example 4 of the present invention regarding hematopoietic stem cells with long-term reconstitution capability expanded by IWR-1-endo, wherein A is a recipient mouse transplanted with DMSO or IWR-1-endo expanded cells, and the human CD45 in its bone marrow... + Cell engraftment level (4 months post-transplantation, n = 10-27), B represents quantitative analysis of human hematopoietic cell subsets in bone marrow, and C represents human CD34 in bone marrow. + CD45 + The presence of hematopoietic stem / progenitor cells is shown in D, which is a representative flow cytometry plot of the engraftment of DMSO expanded cells, IWR-1-endo expanded cells, and bone marrow-derived hematopoietic stem / progenitor cells in the bone marrow of recipient mice 4 months after transplantation. E is the frequency of hematopoietic stem cells in cHSPCs culture, IWR-1-endo cultured cells, and bone marrow-derived hematopoietic stem / progenitor cells, which was determined and calculated by ELDA software. Each dilution has n=5 mice. F shows the engraftment of peripheral blood in recipient mice in the secondary transplantation experiment, n=5-10. Figure 12 This is the second set of IWR-1-endo amplified hematopoietic stem cell data with long-term reconstitution capability provided in Example 4 of the present invention, demonstrating human CD34 in bone marrow. + CD45 + Representative flow cytometry plots of hematopoietic stem / progenitor cells; Figure 13 The IWR-1-endo treatment provided in Example 5 of this invention inhibits the Wnt signaling pathway and upregulates lysosomes and HSC stemness-related genes, wherein A represents the effect of treatment on CD34 cells cultured on day 7. +Bulk RNA-seq principal component analysis of hematopoietic stem / progenitor cells, n=3 for each group; B is the volcano plot of differentially expressed genes (IWR-1-endo group and control group), n=3; C is the heatmap visualization; D is the Western blotting analysis. Figure 14 This is a statistical diagram of the relative mRNA expression levels of Wnt repressor genes related to IWR-1-endo in cHSPC culture group, DMSO-treated co-culture group, or IWR-1-endo-treated co-culture group provided in Example 5 of the present invention (n=3 independent experiments) and qRT-PCR analysis of the differences in hematopoietic stem cell gene expression. A is... NOTCH2 B is JUN C is AXIN D is TANKS E is TANKS2 F is LAMP G is NOTCH H is c-Kit I is HOXB4 J is CTNNBI ; Figure 15 The data provided in Example 6 of this invention, which reveals NISS-driven cellular heterogeneity and IWR-1-endo-mediated state transitions through single-cell transcriptomics, presents UMAP (Uniform Manifold Approximation and Projection) projections of 45,682 single cells from two donors at days 0, 5, and 10 of culture (DMSO vs. IWR-1-endo treatment). Figure 16 This is the second set of single-cell transcriptomics data provided in Example 6 of the present invention to reveal NISS-driven cellular heterogeneity and IWR-1-endo-mediated state transitions. In this data, A represents the expression patterns of key marker genes defining the identity of each cell cluster, including HSC / MPP markers CD34, HOXA9, TAL1, CD117, and HOXB4; B represents the quantitative analysis of the abundance of each cell cluster, with n=2 donors; and C represents the dynamic changes in UMAP according to the treatment group, showing selective enrichment of HSC / MPP cell populations under IWR-1-endo conditions (red density superposition). Figure 17 The single-cell transcriptomics data provided in Example 6 of this invention reveal NISS-driven cellular heterogeneity and IWR-1-endo-mediated state transitions, demonstrating the changes in treatment effects over time. Figure 18The single-cell transcriptomics data provided in Example 6 of this invention reveal NISS-driven cellular heterogeneity and IWR-1-endo-mediated state transitions, where A represents stress responses under different conditions, BC represents changes in the binding signal of β-catenin in the promoter / enhancer region of the SOD2 gene, and D and E represent quantitative analyses showing fold changes in SOD2 mRNA and SOD2 protein levels under different culture conditions. Figure 19 The GSEA analysis results for the synergistic NISS relief effect of Wnt / TGF-β dual inhibition provided in Example 7 of the present invention, n=3; Figure 20 The results of the synergistic NISS relief effect of Wnt / TGF-β dual inhibition provided in Example 7 of this invention, wherein AC represents the inhibition of the expression of TGF-β signaling pathway-related genes (TGFB1, ID2, SMAD7) by IWR-1-endo, n=3, and D represents the frequency of hematopoietic stem / progenitor cells after 7 days of culture (LIN). - CD45RA - CD34 + CD38 low / - Phenotypic fold change, n=3, E and G are the fold changes in the absolute number of hematopoietic stem / progenitor cells at different gene loci, n=3, F is the fold change after in vivo implantation, n=8-12 mice per group; Figure 21 This is the result of Example 7 of the present invention, which uses the limiting dilution method to analyze the effect of combined treatment on stem cell frequency. Figure 22 The second result of the analysis of the effect of combined treatment on stem cell frequency using the limiting dilution method provided in Example 7 of the present invention is as follows: A is the fold change in the number of HSPCs, B is the fold change in the number of in vivo chimeras, C is the logarithmic result of the proportion of non-responders, and D is the confidence interval of the reciprocal of the stem cell frequency in the entire culture system. Detailed Implementation

[0021] In this document, "prepared from" and "comprising" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0022] In this document, the conjunction "composed of" excludes any unspecified elements, steps, or components. If used in a claim, this phrase will make the claim closed, meaning it does not contain any materials other than those described, except for conventional impurities associated with them.

[0023] In this document, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1~5” is disclosed, the described range should be interpreted as including the ranges “1~4”, “1~3”, “1~2”, “1~2 and 4~5”, “1~3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0024] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0025] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbering I...V; A1, A2...A7; first, second...fifth, etc.

[0026] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0027] In this article, “each…independently selected”, “…independently selected respectively”, and “…independently selected” are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The inventors discovered that conventional culture induces Niche-Induced Inflammation Signaling (NISS), which inhibits cHSPC amplification and function, and found that small molecule compounds IWR-1-endo and / or A-83-01 can significantly improve cHSPC amplification and function.

[0030] This invention provides, in one aspect, the application of small molecule compounds in A1 to A4: A1. To prepare products for the expansion of human circulating hematopoietic stem / progenitor cells; A2. To prepare products for improving the regenerative capacity of human circulating hematopoietic stem / progenitor cells; A3. Prepare products for maintaining the stemness of human circulating hematopoietic stem / progenitor cells; A4. Prepare products for hematopoietic reconstitution; The small molecule compounds include IWR-1-endo and / or A-83-01.

[0031] Data show that both small molecule compounds IWR-1-endo and A-83-01 can promote the amplification of cHSPCs. Compared with single reagents, the combination of small molecule compounds IWR-1-endo and A-83-01 has a synergistic promoting effect, significantly promoting the amplification of cHSPCs and maintaining cell stemness and regeneration capacity. This provides a new approach to solving the technical problem that human circulating hematopoietic stem / progenitor cells are difficult to rapidly expand in vitro while maintaining cell stemness and regeneration potential.

[0032] In some specific embodiments, the working concentration of IWR-1-endo is 0.1~10μM; specifically, the working concentration of IWR-1-endo can be, but is not limited to, 0.1μM, 0.5μM, 1μM, 2μM, 4μM, 6μM, 8μM or 10μM, or any value between 0.1 and 10μM.

[0033] In some specific embodiments, the working concentration of A-83-01 is 0.1~5μM. Specifically, the working concentration of A-83-01 can be, but is not limited to, 0.1μM, 0.5μM, 1μM, 2μM, 3μM, 4μM or 5μM, or any value between 0.1 and 5μM.

[0034] In some specific embodiments, the hematopoietic reconstitution includes at least one of treating hematologic malignancies, treating hereditary hematologic diseases, or restoring hematopoietic function after chemotherapy and / or radiotherapy.

[0035] In some specific embodiments, the product for hematopoietic reconstitution comprises a population of human circulating hematopoietic stem / progenitor cells obtained by amplification of human circulating hematopoietic stem / progenitor cells.

[0036] According to another aspect of the invention, a composition for in vitro expansion of human circulating hematopoietic stem / progenitor cells is also provided, comprising IWR-1-endo and / or A-83-01.

[0037] In some specific embodiments, the final concentration of IWR-1-endo is 0.1 ~ 10 μM; in some specific embodiments, the final concentration of A-83-01 is 0.1 ~ 5 μM.

[0038] According to another aspect of the present invention, a culture medium for in vitro expansion of human circulating hematopoietic stem / progenitor cells is also provided, comprising the above-described composition.

[0039] In some specific embodiments, the culture medium further includes a basal culture medium and hematopoietic support factors; in some specific embodiments, the hematopoietic support factors include SCF, TPO, FLT3L, IL-3, and IL-6; in some specific embodiments, the working concentrations of SCF, TPO, and FLT3L are each independently 50-100 ng / mL; in some specific embodiments, the working concentrations of IL-3 and IL-6 are each independently 5-20 ng / mL.

[0040] According to another aspect of the present invention, a method for in vitro expansion of human circulating hematopoietic stem / progenitor cells for non-disease diagnosis and treatment purposes is also provided, comprising culturing peripheral blood mononuclear cells (PBMCs) containing cHSPCs using the culture medium described above.

[0041] In some specific implementations, the culture time is 5 to 10 days; in some specific implementations, the culture medium is replaced every 2 to 3 days during the culture period.

[0042] According to another aspect of the present invention, a human circulating hematopoietic stem / progenitor cell population is also provided, which is obtained by the above-described method.

[0043] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0044] Materials and Methods 1. Human CD34 + Cell culture Human CD34 +Cells were cultured in hematopoietic stem cell expansion medium consisting of StemSpanSFEM II (StemCell Technologies, catalog number: 09655) supplemented with the following factors: 100 ng / ml human stem cell factor (SCF), 100 ng / ml human FMS-like tyrosine kinase 3 ligand (FLT3L), 50 ng / ml human thrombopoietin (TPO), and 1 µM SR1. The compounds were added to the HSC expansion medium at specified concentrations.

[0045] 2. Community Forming Unit (CFU) Experiment CD34 from fresh bone marrow + CD34 cells or CD34 cells cultured under different conditions + Cells were resuspended in 200 μl of IMDM (Thermo Fisher Scientific, 12440061) containing 2% FBS (Hyclone, catalog number: SH30396) and then seeded into 2 ml of methylcellulose medium (MethoCult™ H4435 Enriched, STEMCELLS Technologies, catalog number: 04435), seeding 1000 or 5000 cells per well as directed, using ultra-low adsorption six-well plates (Corning, catalog number: 3471). The plates were incubated at 37°C in a 5% CO2 incubator. After 14 days, the number of different CFU types was counted, including granulocyte CFU (CFU-G), macrophage CFU (CFU-M), granulocyte-macrophage CFU (CFU-GM), erythrocyte CFU (CFU-E), erythrocyte bursting unit (BFU-E), and granulocyte, erythrocyte, macrophage, and megakaryocyte CFU (CFU-GEMM). Each experiment has three duplicate wells.

[0046] 3. Flow cytometry analysis Cultured cells were collected at different time points and incubated with the specified antibody in PBS containing 0.5% BSA (Sigma, catalog number: A1470-100G) at 4°C for 30 minutes. Subsequently, the cells were washed three times with PBS and resuspended in 0.2 ml PBS for analysis. Flow cytometry analysis was performed using a CytoFLEX (BECKMAN COULTER) instrument. Data were analyzed using FlowJo-V10 (BD). The antibodies used were: anti-human CD34, anti-human CD38, anti-human CD49f, anti-human CD90, and anti-human CD45RA. For analysis of post-transplant hematopoietic lineage, peripheral blood cells were collected at specified time points. Bone marrow cells were isolated 4 months post-transplant and stained with the following antibodies: anti-human CD45, anti-mouse CD45, anti-human CD19, anti-human CD15, anti-human CD3, anti-human CD4, and anti-human CD8.

[0047] 4. Monitoring of human chimerism in primary transplantation and NCG mice NCG mice were irradiated with a sublethal dose 4–6 hours prior to transplantation. At designated post-transplantation time points, the reconstituted human cells in peripheral blood and bone marrow of NCG mice were monitored by flow cytometry. Four months post-transplantation, bone marrow cells from both femurs were collected. The proportion of reconstituted human cells in peripheral blood was assessed at 1, 2, 3, and 4 months post-transplantation, and analyzed according to the flow cytometry protocol. Cells were washed with PBS containing 0.5% BSA, and chimerism was analyzed using anti-human CD45 and anti-mouse CD45. Flow cytometry analysis was performed using CytoFLEX. The correspondence between antibody markers and cell populations is as follows: CD15 (myeloid cells); CD19 (B lymphocytes); CD3 (T lymphocytes).

[0048] 5. Secondary transplantation and in vivo human cell monitoring Mice with average chimerism efficiency from primary transplantation were selected from each group (drug-treated group and DMSO-amplified cell group) for secondary transplantation. These mice were sacrificed, and femurs and tibias were collected. Bone marrow was flushed out with PBS containing 0.5% BSA. Bone marrow cells were pipetted into a single-cell suspension. The suspension was then processed at a rate of 1×10-1. 7 Cells per mouse were transplanted. To assess the reconstitution efficiency of the secondary graft, peripheral blood from mice undergoing secondary transplantation was periodically analyzed post-transplantation. Freshly collected cells were analyzed by flow cytometry. Cells were washed with PBS containing 0.5% BSA, and chimerism was analyzed using anti-human CD45 and anti-mouse CD45. Flow cytometry analysis was performed using CytoFLEX. The correspondence between antibody markers and cell populations is as follows: CD15 (myeloid cells); CD19 (B lymphocytes); CD3 (T lymphocytes).

[0049] 6. Limiting dilution analysis HSC frequencies were quantified using extreme dilution analysis (http: / / bioinf.wehi.edu.au / software / elda / ) and 95% confidence intervals were calculated. In this invention, all CD45 groups in the recipient's bone marrow after transplantation were analyzed. + Human CD45 in cells + A proportion >0.1% is defined as positive chimerism. This standard applies to LDA calculations for both primary and secondary transplants.

[0050] 7. Western Blot 1×10 6 A fresh mononuclear cell PBMC (containing CD34) + Cells were co-cultured with the small molecule compound IWR-1-endo (1 μM) and / or A-83-01 (5 μM) or DMSO (0.01% v / v) for 7–10 days, then centrifuged and washed with PBS. The pellet was resuspended in 45 μl PBS and 15 μl lysis buffer (200 mM Tris-HCl, 8% SDS, 400 mM DTT, 0.1% bromophenol blue, 40% glycerol) and incubated at 100°C for 10 min for lysis. The lysis buffer was electrophoresed on a 10% SDS-PAGE gel and then transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with an appropriate primary antibody in 4% skim milk. It was then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse / rabbit IgG (Jackson Laboratories) secondary antibody, washed, and visualized using a Western blot imaging system.

[0051] 8. RNA-seq library construction and analysis RNA was isolated from the target cells. During RNA extraction and quality control, the manufacturer's operating procedures were strictly followed. Total RNA was extracted using the RNeasy kit (Kiagen, Germany) combined with on-column DNase I digestion (Kiagen, Germany). RNA concentration and integrity were detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was performed using iScript reverse transcription ultramixed buffer (Bio-Rad Laboratories, USA).

[0052] The generated sequencing reads were aligned to the GRCh38 version of the human genome using the TopHat alignment software tool. The expression values ​​for each sample were calculated using cuffquant by reading the aligned BAM file. Then, cuffnorm was used to merge the expression values ​​into a single FPKM table.

[0053] After sequencing library construction, 150 bp paired-end sequencing (NovaSeq-PE150) was performed using the Illumina HiSeq platform (Illumina Corporation, USA). The raw sequencing data has been stored at the National Center for Biotechnology Information (accession number: HRA015560) and made publicly available through the genome sequence archive database (https: / / ngdc.cncb.ac.cn / gsa-human).

[0054] The bioinformatics analysis process is as follows: read processing, using HISAT2 to align the original reads to the human reference genome (GRCh38); gene quantification, using HTSeq (v0.6.0) to calculate gene level counts; after calculating FPKM (number of mapped fragments per kilobase transcript), differentially expressed gene analysis was performed using (22) (1.10.1), and the Benjamini-Hochberg method was used to correct the P-value to control the false discovery rate. Genes with a corrected P-value < 0.05 after DESeq2 calculation were defined as differentially expressed genes.

[0055] Principal component analysis based on FPKM values ​​was performed to reveal the expression pattern characteristics between groups. Enrichment analysis of gene ontology and the Kyoto Encyclopedia of Genes and Genomes was conducted using the clusterProfiler R package (version 4.12.0) (23), DAVID Bioinformatics Resources (http: / / www.david.ncifcrf.org / ), or the KOBAS platform (http: / / bioinfo.org / kobas / ). A corrected P-value < 0.05 was considered significant enrichment.

[0056] 9. Quantitative and Statistical Analysis Data are expressed as mean ± standard error (SEM) or standard deviation (SD). Paired comparisons between different groups were assessed using unpaired t-tests. For all analyses, p < 0.05 was considered statistically significant. Statistical significance and n-values ​​are reported in the figure captions.

[0057] All data are presented as mean ± standard error. Statistical significance was assessed using an unpaired t-test, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0058] 10. Reagents A-83-01: Purchased from MedChemExpress (MCE), lot number HY-10432; IWR-1-endo: Purchased from MedChemExpress (MCE), lot number HY-12238.

[0059] Example 1: Establishment and validation of an in vitro cHSPC culture system and induction of niche-induced inflammatory signaling (NISS) in the microenvironment. To investigate the potential and related obstacles of in vitro expansion of cHSPCs, this embodiment first established a co-culture system simulating the in vivo microenvironment. For example... Figure 1 As shown, culturing cHSPCs alone or co-culturing them with PBMC helper cells in standard cytokine-containing medium (SCF, TPO, FLT3L, IL-3, IL-6, SR1) leads to rapid activation of inflammatory factors in both cHSPCs and helper cells; this process is termed niche-induced inflammatory signaling network. CD34 was isolated from peripheral blood of healthy donors. + cHSPCs (approximately ~0.01-0.1% of PBMCs). To simulate cell-cell interactions, the culture included PBMCs after cHSPC removal (in... Figure 1 (Represented as PBMC helper cells). After 7 days of culture alone with standard cytokine therapy including SCF, FLT3L, TPO, IL-3, IL-6, and the small molecule StemRegenin1 (SR1), cHSPCs showed the following results: Figure 2 As shown in Figure A, despite the support of cytokines, LIN CD45RA CD34 + CD38low / cHSPCs still showed a significant decrease of 11.09 ± 4.83-fold, with a loss of over 89% of the original phenotype by day 7. cHSPCs exhibited limited expansion and stem / progenitor cell frequency (defined as Lin). CD45RA CD34 + CD38 The significant decrease in cells indicates the existence of a mechanism that inhibits stemness. Figure 2 The B-cell model shows that, with the support of helper cells, LIN CD45RA CD34 + CD38low / HSPCs achieved a significant improvement of 13.55 ± 3.00-fold by day 7. Co-culture of cHSPCs with PBMC helper cells promoted HSPC expansion to some extent, but the expansion efficiency was still limited.

[0060] Comparative transcriptomic analysis of cHSPCs cultured alone and cHSPCs co-cultured with PBMC helper cells revealed inflammatory response characteristics in the co-culture group, such as... Figure 3As shown. The inflammatory response characteristics in the co-culture group were defined as microenvironment-induced inflammatory stress signals (NISS). For example... Figure 3 As shown in Figure A, KEGG pathway enrichment analysis revealed that cultured cHSPCs were significantly enriched in inflammation, cell cycle, and differentiation-related signaling pathways, including cytokine-cytokine receptor interactions, the JAK-STAT signaling pathway, Th17 cell differentiation, and the p53 signaling pathway, but not in pathways related to stemness maintenance. This indicates that in vitro culture conditions actively induced transcriptional programs for inflammation, differentiation, and apoptosis. Figure 3 Gene interaction network analysis using significantly upregulated genes in individual cHSPC cultures in media B and C showed that these inflammatory factors (IFNG, IRF4, TNF superfamily) are closely related to cell cycle (CDKN2A, CCND2), interleukins (IL2, IL9, IL17A) and hematopoietic differentiation (CD19, CD70, CD40L).

[0061] To assess the impact of NISS on long-term implantation function, an in vivo transplantation experiment was conducted. Results are as follows: Figure 4 As shown, in xenograft experiments, cHSPCs cultured alone exhibited extremely low chimerism (<0.1% at 4 months). cHSPCs co-cultured with PBMC helper cells achieved multilineage engraftment, but the level of chimerism remained limited. This indicates that cHSPCs cultured alone completely lost their in vivo engraftment capability. Conversely, when co-cultured with PBMC helper cells, the expanded cells retained some engraftment activity, but the level of chimerism remained significantly limited.

[0062] This embodiment established an in vitro culture system to investigate barriers to cHSPC expansion. Using this system, it was found that NISS, an inflammatory stress program activated in individually cultured cHSPCs, is a key obstacle impairing their expansion and implantation functions. Although co-culturing with PBMC helper cells can partially alleviate NISS and preserve some stem cell function, it still cannot fully unlock the potential of cHSPCs.

[0063] Example 2: Screening of small molecule compounds that effectively amplify cHSPCs To identify small molecule compounds that can effectively amplify cHSPCs, preliminary screening was conducted based on the influence of candidate compounds on the total number of nucleated cells (TNCs) during cHSPC amplification. For example... Figure 5As shown, cHSPCs were co-cultured with peripheral blood mononuclear helper cells in StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L, IL-3, IL-6, and SR1, and small molecule compounds to be screened were added. The total number of nucleated cells was then dynamically monitored, and effective compounds were selected for further functional validation. The specific effects of the small molecule compounds to be screened on TNCs in the co-culture of cHSPCs and PBMCs were monitored. The results are as follows: Figure 6 As shown, compounds IWR-1-endo and A-83-01 exhibited a clear effect on the total number of nucleated cells, indicating that the Wnt pathway inhibitor IWR-1-endo had the most significant effect on TNC amplification, followed by the TGF-β pathway inhibitor A-83-01.

[0064] The ability of IWR-1-endo to inhibit NISS was then examined. Figure 7 Transcriptome sequencing revealed that, in addition to the expected inhibition of the Wnt pathway, IWR-1-endo treatment significantly downregulated several key inflammatory signaling pathways, including NF-κB, IL-17, TNF, and Toll-like receptor signaling pathways. Furthermore, Figure 8 The results showed that IWR-1-endo treatment significantly upregulated the stemness-related gene CD34, while downregulating the expression of inflammatory factors / differentiation genes such as IFNG, IRF8, CD79B and CD2 at the mRNA or protein level.

[0065] The above results indicate that IWR-1-endo suppresses inflammatory signaling and promotes stem cell-like states by upregulating CD34 and simultaneously inhibiting differentiation-related genes, making it a potential target small molecule for amplifying cHSPCs.

[0066] Example 3: IWR-1-endo treatment significantly improved cHSPC amplification and its function. This embodiment investigates whether IWR-1-endo can effectively promote cHSPC amplification and its function, as well as the potential mechanism. The experimental procedure is illustrated in the diagram below. Figure 9 As shown in Figure A.

[0067] Based on the screening results of Example 2, Wnt pathway inhibitors were further refined, including Porcupine inhibitors (LGK974, ETC-159, IWP-2, IWP-4), LRP5 / 6 inhibitors (sclerostin and DKK1), AXIN stabilizer (XAV939), TCF / β-catenin interaction inhibitor (PKF118-310), and β-catenin degradation inducer PRI-724. Two concentrations of the above-mentioned small molecule compounds were used to elucidate the precise role of Wnt signaling in regulating cHSPC amplification.

[0068] The expression of TNCs and HSPC surface markers LIN, CD45RA, CD34, and CD38 was used as an efficiency indicator. Figure 9 As shown in Figure B, during the culture and screening process, all ten compounds were observed to significantly increase LIN levels at both test concentrations. CD45RA CD34 + CD38low / Frequency and quantity of HSPCs (fold change relative to DMSO > 1.1). IWR-1-endo significantly increased LIN levels compared to the DMSO-treated control group after addition of the medium. CD45RA CD34 + CD38low / The frequency and quantity of HSPCs were investigated. IWR-1-endo showed the best effect, so the influence of IWR-1-endo on the in vitro and in vivo amplification of cHSPCs was further studied.

[0069] like Figure 10 As shown in Figure A, the effects of different doses of IWR-1-endo on TNCs and LIN were tested. CD45RA CD34 + CD38 low / - Effects on subpopulation expansion. The highest expansion levels of these cells were observed under treatment with 1 μM IWR-1-endo. Treatment with IWR-1-endo in addition to cytokines led to LIN... CD45RA CD34 + CD38 low / The amplification of HSPCs was significantly enhanced, exceeding that of the control group (cytokines plus 0.1% DMSO) by 6.22 ± 2.78 times. These results indicate that IWR-1-endo can effectively amplify cHSPCs in vitro.

[0070] like Figure 10 As shown in Figures B and C, colony-forming unit (CFU) experiments were performed to determine the pluripotency of cells expanded by IWR-1-endo. The results showed that IWR-1-endo increased the number of granulocyte, erythrocyte, macrophage, and megakaryocyte colony-forming units (CFU-GEMM) by approximately fourfold, while the number of other types of CFU, including erythrocyte colony-forming units (CFU-Es) or granulocyte and macrophage colony-forming units (CFU-GMs), was also significantly affected by IWR-1-endo treatment.

[0071] The above results indicate that IWR-1-endo effectively promotes the amplification of cHSPCs with multi-directional differentiation potential.

[0072] Example 4: IWR-1-endo amplified cHSPCs exhibit long-term reconstruction capability This example investigated whether IWR-1-endo expanded cells possess the ability to be reconstituted in vivo over a long period.

[0073] 2×10 6 IWR-1-endo treated cells cultured for 7-10 days (corresponding to 3×10⁶ cells) 4 LIN on day 0 CD45RA CD34 + CD38 low / - cHSPCs or DMSO-treated cells were injected into immunodeficient mice, and then human CD45 reconstituted in the peripheral blood of recipient mice was quantified at 1, 2, 3, and 4 months post-transplantation. + Cell frequency. For example... Figure 10 As shown in Figure D, IWR-1-endo expanded cells exhibited a continuously increasing engraftment rate in peripheral blood at all time points. Specifically, the chimerism rate achieved by IWR-1-endo expanded cells at 4 months was 18.25 times that of the DMSO control group (P<0.0001). These data indicate that IWR-1-endo treatment significantly enhances the engraftment capacity of expanded cHSPCs.

[0074] Hematopoietic output in the peripheral blood of recipient mice at 1, 2, 3, and 4 months post-transplantation was analyzed to assess whether IWR-1-endo expanded cells retained multilineage differentiation potential. Figure 10 As shown in Figure E, compared with control cells, IWR-1-endo treated cells exhibited multi-lineage reconstitution potential and were able to generate myeloid (CD15) lineages in vivo. + B lymphatic system (CD19) + ) cells, T lymphocytes (CD3) + ) cells, helper T cells (CD3) + CD4 + ) and cytotoxic T cells (CD3) + CD8 + The results showed that the human grafts of IWR-1-endo expanded cells were mainly composed of lymphoid and myeloid cells, and were similar to mobilized bone marrow-derived CD34 cells. + Cellular similarity.

[0075] Four months post-transplantation, human chimerism in the bone marrow of recipient mice was assessed, and the results were as follows: Figure 11The A. IWR-1-endo treatment group showed higher levels of human CD45 than other groups. + Cell chimerism rate. For example... Figure 11 The distribution of hematopoietic lineages in the middle class, including myeloid, B-cell, and T-cell lineages, was comparable to that of the BM-HSPC positive control group. Figure 11 In C, human CD34 was detected in the bone marrow. + CD45 + Implantation of HSPCs. For example... Figure 11 China D and Figure 12 The representative flow cytometry plots describe the human CD45 at this time. + Chimerism of cells, various hematopoietic cells, and human HSPCs.

[0076] The frequency of HSCs after IWR-1-endo treatment was determined by extreme dilution assay (ELDA). 6 10 5 and 10 4 1 cell (adjusted to day 0 CD34) + Cells with numbers of approximately 15,000, 1,500, and 150 were transplanted into immunodeficient NCG mice. Four weeks post-transplantation, analysis showed that the positive control group mobilized bone marrow-derived CD34 cells... + The HSC frequency of the cells was 1 / 2,042, the cHSPC amplification group co-cultured with helper cells had a frequency of 1 / 26,114, and the IWR-1-endo amplification group had a significantly higher frequency of 1 / 835. Figure 11 The presence of E in the image indicates that the reconstructed HSPCs were significantly amplified during culture using IWR-1-endo.

[0077] This study determined whether IWR-1-endo expanded cells could maintain continuous engraftment through secondary transplantation using bone marrow cells isolated from primary recipient mice injected with either IWR-1-endo expanded cells or BM-HSPCs four months post-transplantation. These cells successfully established human engraftment in secondary recipients, such as... Figure 11 The level of F in peripheral blood was similar to that in the positive control group, indicating that long-term HSCs (LT-HSCs) were expanded in IWR-1-endo treated cells.

[0078] The above results indicate that IWR-1-endo expanded cells retain the ability to form multilineage blood cells and exhibit long-term engraftment capability.

[0079] Example 5: IWR-1-endo treatment inhibits Wnt signal expression This embodiment compared the expression of Wnt signaling pathway-related genes in cHSPCs amplified by IWR-1-endo, control cHSPCs cultured alone, and cHSPCs co-cultured with PBMC helper cells to determine the mechanism by which IWR-1-endo promotes the amplification of human cHSPCs.

[0080] like Figure 13 In the middle A, principal component analysis (PCA) of the transcriptome showed that the samples from different treatment groups exhibited significant clustering, with the IWR-1-endo treatment group and the CD34-only group showing the most clustering. + The control and DMSO-treated co-culture samples clustered together. This clear separation is consistent with the significant whole-transcriptome differences induced by different culture conditions, while the tight clustering within groups reflects the high similarity of transcriptome profiles among repeated samples under the same conditions.

[0081] like Figure 13 Volcano plot analysis in the DMSO control group revealed distinct transcriptional profiles between the control groups. Genes upregulated by IWR-1-endo treatment involved metabolism (FBP1, G0S2, CES1, HSD11B1, CP), immune and inflammatory responses (MARCO, ORM1, GPNMB), stress responses (NUPR1, ORM1, CP), and drug detoxification (SULT1C2, CES1). However, the top 10 upregulated genes in the DMSO control group highlighted B cell biological signatures (e.g., FCRL5, BANK1, MS4A1 / CD20), Th1 / Th17 cytokines (IFNG, IL17F), interferon signaling (IFI44L), and epigenetics (e.g., histone methyltransferase KMT2A).

[0082] like Figure 13 The heatmap of gene expression in the key Wnt pathway showed that IWR-1-endo treatment significantly suppressed the expression of Wnt signaling components.

[0083] like Figure 13 China D and Figure 14 The study confirmed the decreased expression of the key Wnt pathway gene CTNNB1 at both the mRNA and protein levels. Figure 14 In both A and B, other key genes in the Wnt pathway, including NOTCH2 and JUN, were downregulated, while Wnt pathway inhibitors such as AXIN, TANK, and TANK2 were upregulated (e.g., ...). Figure 14 (CE). Furthermore, through quantitative real-time PCR experiments, lysosomal gene LAMP (such as...) Figure 14 (F) and HSC stem genes such as NOTCH, c-Kit and HOXB4 (e.g.) Figure 14 The GI (medium glutamate) was also upregulated due to IWR-1-endo treatment.

[0084] The data above indicate that IWR-1-endo inhibits the Wnt pathway while upregulating lysosomes and stemness-related genes.

[0085] Example 6: Single-cell RNA sequencing elucidates that IWR-1-endo suppresses HSPC culture-induced inflammatory state through transcriptional reprogramming. Example 1 established an in vivo simulation system of cHSPC plus PBMC helper cell co-culture, which showed significant advantages over cHSPC culture alone, but still induced NISS and significantly inhibited cHSPC amplification and function. Therefore, this example further investigated the exact nature of the NISS state and the mechanism by which IWR-1-endo overcomes it. To this end, single-cell RNA sequencing (scRNA-seq) was performed on the co-culture systems with and without IWR-1-endo treatment. The status of PBMCs from the two donors on days 0, 5, and 10 was applied and analyzed. BM-HSPCs were used as a positive control.

[0086] like Figure 15 scRNA-seq analysis revealed 15 distinct cellular types at the transcriptional level, including a well-defined HSC / MPP population, each defined by a unique set of marker genes (e.g., Figure 16 (A) For example Figure 16 In the B-cell analysis, cluster abundance quantification showed that IWR-1-endo treatment significantly altered cellular hierarchical structure, particularly driving the expansion of HSCs / MPPs. Figure 16 In the UMAP plot, this effect can be directly observed; the cell density in the HSC / MPP region increased significantly after treatment, confirming the amplifying effect of IWR-1-endo on this key original population. Transcriptional dynamics from both donors showed similar trends throughout culture, indicating the robustness of this culture system in amplifying HSCs / MPPs.

[0087] like Figure 17 UMAP analysis revealed that IWR-1-endo treatment profoundly reshaped the cellular state. On day 0, cells occupied a distinct region, while co-cultured cells were localized in a separate cluster. After IWR-1-endo treatment, the cell population exhibited complete spatial separation in UMAP space, indicating that IWR-1-endo significantly altered the cellular transcriptional profile.

[0088] Notably, inflammation-related pathways, including neutrophil degranulation, platelet activation signaling, and aggregation, were significantly downregulated after IWR-1-endo treatment. Conversely, pathways such as glycolysis and telomere maintenance were significantly upregulated. Figure 18In the middle A, the cellular response to stress was also significantly enhanced after IWR-1-endo treatment.

[0089] To investigate the mechanism by which IWR-1-endo upregulates stress response genes, cut & tag analysis was performed on β-catenin. The analysis showed that β-catenin binding was enriched at stress response gene sites in the IWR-1-endo treated group. Cross-referencing with transcriptome data confirmed that most of these genes were also significantly upregulated at the mRNA level. Notably, superoxide dismutase 2 (SOD2) emerged as a gene of particular interest. SOD2 is a key mitochondrial antioxidant enzyme that catalyzes the conversion of superoxide radicals into hydrogen peroxide and oxygen, playing a central role in maintaining redox homeostasis and protecting mitochondrial components from oxidative damage. Further analysis indicated that IWR-1-endo treatment significantly enhanced β-catenin binding at SOD2 gene sites (e.g., Figure 18 (BC), accompanied by consistent upregulation of SOD2 at both the transcriptomic and proteomic levels (e.g., Figure 18 middle DE).

[0090] Example 7: Dual Inhibition of Wnt and TGF-β by NISS Unleashes cHSPC Potential like Figure 19 Transcriptome analysis showed that the TGF-β signaling pathway was enriched in the DMSO control group compared to the IWR-1-endo treatment group (NES = 1.76, FDR = 0.031). In cHSPCs co-cultured with PBMC helper cells, TGF-β signaling was significantly activated, suggesting it may constitute a different major parallel signaling axis than NISS. Key effectors, including TGFB1, ID2, and SMAD7, were upregulated (e.g., Figure 20 (AC). This makes TGF-β an auxiliary signal axis for inhibiting cHSPC amplification and function of NISS.

[0091] β-catenin CUT & Tag analysis. Results showed that β-catenin can directly bind to the gene promoter regions of TCF3 and SKI, key regulators of the TGF-β pathway. Figure 20 DG (China)

[0092] Small molecule screening in Example 2 showed that the TGF-β signaling inhibitor A-83-01 increased TNCs in cHSPCs co-cultured with PBMC helper cells. Based on these results, the potential of A-83-01 treatment was further examined to determine whether it could further unlock the potential of cHSPCs. As a monotherapy, inhibition of TGF-β alone increased LIN... CD45RA CD34+ CD38 low / + The frequency and number of HSPCs increased by nearly 5 times, indicating that the TGF-β signal independently limited cHSPC amplification.

[0093] The dual treatment with A-83-01 and IWR-1-endo produced a profound synergistic effect. For example... Figure 21 As shown, the HSPC frequency (LIN) after 7 days of culture. - CD45RA - CD34 + CD38 low / - Differences in changes were observed. A-83-01 alone increased HSPC frequency by 4.9-fold; IWR-1-endo alone by 6.2-fold; and the combination group by 9.11-fold (P < 0.0001 compared to either single-drug group). n = 3. Figure 22 In group A, the total number of HSPCs increased by 11.99 ± 3.4 times compared to the co-culture control group treated with DMSO, demonstrating a synergistic effect in relieving NISS (Bliss independence score of 1.68). n=3.

[0094] like Figure 22 The study showed a synergistic effect in in vivo transplantation. Four months post-transplantation, the chimerism rate of cells expanded by the combined treatment in peripheral blood was 2.11 times higher than that of the A-83-01 group alone and 1.56 times higher than that of the IWR-1-endo group alone. Each group consisted of 8–12 mice. This functional synergy was demonstrated in HSPC amplification frequency, yield, and reconstitution capacity, establishing A-83-01 / IWR-1-endo as an effective combination for enhancing cHSPC amplification and function.

[0095] like Figure 22 In C and D tests, LDA assays showed that the combination treatment significantly increased the frequency of functional stem cells. The stem cell frequency obtained using the combination of IWR-1-endo and A-83-01 was 1 / 797, while the frequency in the A-83-01-only treatment group was 1 / 1330. Compared with the stem cell frequency of IWR-1-endo alone in Example 4 (1 / 835), the results indicate that the drug combination significantly promoted stem cell frequency. The combination of IWR-1-endo and A-83-01 produced a synergistic effect, comprehensively alleviating NISS, thereby increasing the quantity and quality of hematopoietic stem / progenitor cells.

[0096] The data above indicate that NISS-induced signaling, including the NF-κB, JUN, Wnt, and TGF-β cascade reactions, constitutes the molecular brake on the cHSPC potential. Releasing these pathways can release the amplification and reconstruction capabilities of human cHSPCs.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Applications of small molecule compounds in A1~A4: A1. To prepare products for the expansion of human circulating hematopoietic stem / progenitor cells; A2. To prepare products for improving the regenerative capacity of human circulating hematopoietic stem / progenitor cells; A3. Prepare products for maintaining the stemness of human circulating hematopoietic stem / progenitor cells; A4. Prepare products for hematopoietic reconstitution; The small molecule compounds include IWR-1-endo and / or A-83-01.

2. The application according to claim 1, characterized in that, The working concentration of the IWR-1-endo is 0.1~10 μM.

3. The application according to claim 1, characterized in that, The working concentration of A-83-01 is 0.1~5μM.

4. The application according to claim 1, characterized in that, The hematopoietic reconstitution includes at least one of B1 to B3: B1. Treatment of hematologic malignancies; B2. Treatment of hereditary blood diseases; B3. Restore hematopoietic function after chemotherapy and / or radiotherapy; The product for hematopoietic reconstitution comprises a population of human circulating hematopoietic stem / progenitor cells obtained by amplification of human circulating hematopoietic stem / progenitor cells.

5. A composition for in vitro expansion of human circulating hematopoietic stem / progenitor cells, characterized in that, Including IWR-1-endo and / or A-83-01.

6. The composition according to claim 5, characterized in that, The final concentration of the IWR-1-endo is 0.1~10 μM; The final concentration of A-83-01 is 0.1~5μM.

7. A culture medium for in vitro expansion of human circulating hematopoietic stem / progenitor cells, characterized in that, Includes the composition described in claim 5 or 6.

8. The culture medium according to claim 7, characterized in that, The culture medium also includes basal culture medium and hematopoietic support factors; The hematopoietic support factors include SCF, TPO, FLT3L, IL-3, and IL-6; The working concentrations of SCF, TPO, and FLT3L are each independently 50–100 ng / mL; The working concentrations of IL-3 and IL-6 are independently 5~20 ng / mL.

9. A method for in vitro expansion of human circulating hematopoietic stem / progenitor cells for non-disease diagnosis and treatment purposes, characterized in that, This includes culturing peripheral blood mononuclear cells containing cHSPCs using the culture medium described in claim 7 or 8; The cultivation time is 5-10 days; Replace the culture medium every 2-3 days during the culture period.

10. A human circulating hematopoietic stem / progenitor cell population, characterized in that, It is amplified by the method described in claim 9.

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