Use of phloroglucinol in the expansion and / or functional enhancement of human circulating hematopoietic stem / progenitor cells

Abstract

This invention provides the application of phloroglucinol in the expansion and / or functional enhancement of human circulating hematopoietic stem / progenitor cells, relating to the fields of stem cell biology and regenerative medicine. Phloroglucinol possesses dual inhibitory capabilities against both NF-κB and JNK / c-Jun, effectively reducing the protein level of phosphorylated NF-κB p65 (Ser536) in cultured cells, accompanied by upregulation of HOXB4 gene expression, and significantly improving LIN... ‑ CD45RA ‑ CD34 + CD38 low / ‑ The expansion fold of cHSPCs cells, maintaining cell stemness and regeneration capacity, provides 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.

Description

Technical Field

[0001] This invention relates to the fields of stem cell biology and regenerative medicine, and in particular to the application of phloroglucinol in the expansion and / or functional enhancement 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] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of phloroglucinol in the expansion and / or functional 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.

[0006] 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 phloroglucinol 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 reconstruction.

[0007] Furthermore, the working concentration of the phloroglucinol is 10~100μM, preferably 20μM.

[0008] 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.

[0009] In a second aspect, the present invention provides a composition for in vitro amplification of human circulating hematopoietic stem / progenitor cells, comprising phloroglucinol.

[0010] Furthermore, the final concentration of the phloroglucinol is 10~100μM, preferably 20μM.

[0011] Thirdly, the present invention provides a culture medium for in vitro expansion of human circulating hematopoietic stem / progenitor cells, comprising the above-described composition.

[0012] Furthermore, the culture medium also includes basal culture medium and hematopoietic support factors.

[0013] Furthermore, the hematopoietic support factors include SCF, TPO, FLT3L, IL-3, and IL-6.

[0014] Fourthly, the present invention provides a method for in vitro expansion of human circulating hematopoietic stem / progenitor cells for purposes other than disease diagnosis and treatment, comprising culturing peripheral blood mononuclear cells containing cHSPCs using the above-mentioned culture medium.

[0015] Fifthly, the present invention provides a human circulating hematopoietic stem / progenitor cell population, which is obtained by the above-described method.

[0016] The present invention further provides the application of phloroglucinol in the expansion and / or functional enhancement of human circulating hematopoietic stem / progenitor cells. Phloroglucinol possesses dual inhibitory capabilities against both NF-κB and JNK / c-Jun, producing a synergistic effect. It effectively reduces the protein level of phosphorylated NF-κB p65 (Ser536) in cultured cells, accompanied by upregulation of HOXB4 gene expression, significantly improving LIN... - CD45RA - CD34 + CD38 low / -The expansion of cHSPCs promotes clonogenic capacity and maintains cell stemness and regeneration capacity, 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

[0017] 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.

[0018] 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 2 The 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 the proportion of cHSPCs, where A represents cHSPC culture alone and B represents cHSPC co-culture with PBMC helper cells. The data are shown 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, where A is the top KEGG pathway, B is the c-upregulated inflammatory factor interaction network, and C is the comparison of the expression levels of some factors occupying major 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 The phloroglucinol treatment provided in Example 2 of this invention for LIN - CD45RA - CD34 + CD38 low / - Detection results of cHSPCs amplification; Figure 6The resorcinol treatment provided in Example 3 of this invention significantly inhibited the Western blot analysis results of NF-κB p65, phosphorylated p65 (Ser536), c-Jun and phosphorylated c-Jun (Ser63), wherein ACTIN was used as an internal control. Figure 7 The effect of phloroglucinol treatment on the expression level of the stemness gene HOXB4 provided in Example 4 of this invention; Figure 8 The effect of phloroglucinol on the clonogenic ability of hematopoietic stem / progenitor cells provided in Example 5 of this invention. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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).

[0024] 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.

[0025] 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.

[0026] 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.

[0027] This invention provides, in one aspect, the application of phloroglucinol 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 reconstruction.

[0028] The inventors discovered that conventional culture induces Niche-Induced Inflammation Signaling (NISS), which inhibits cHSPC amplification and function. Phloroglucinol possesses dual inhibitory capabilities against both NF-κB and JNK / c-Jun, producing a synergistic effect and more comprehensively blocking the NISS signaling network. Besides inhibiting inflammatory signals, its antioxidant capacity directly targets stress sources, providing a unique protective dimension against cHSPC amplification. It effectively reduces the protein level of phosphorylated NF-κB p65 (Ser536) in cultured cells, accompanied by upregulation of HOXB4 gene expression, and significantly improves LIN... - CD45RA - CD34 + CD38 low / - The cell expansion factor promotes cell clone formation potential, maintains cell stemness and regeneration capacity, and 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.

[0029] In addition, phloroglucinol is a natural compound that may have a better biocompatibility and safety profile, providing a candidate for the development of clinically applicable serum-free or animal-free culture additives.

[0030] In some specific embodiments, the working concentration of phloroglucinol is 10~100μM; specifically, the working concentration of phloroglucinol can be, but is not limited to, 10μM, 20μM, 50μM, 100μM, or any value between 10 and 100μM, preferably 20μM.

[0031] 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.

[0032] 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.

[0033] According to another aspect of the present invention, a composition for in vitro amplification of human circulating hematopoietic stem / progenitor cells, comprising phloroglucinol, is also provided.

[0034] In some specific embodiments, the final concentration of the phloroglucinol is 10~100μM, preferably 20μM.

[0035] 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.

[0036] 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.

[0037] 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 each independently 5-20 ng / mL.

[0038] 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 containing cHSPCs using the above-described culture medium.

[0039] The culture time is 5-10 days; during the culture period, the culture medium can be replaced as needed.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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), with 300 cells per well as directed, using ultra-low adsorption six-well plates (Corning, catalog number: 3471). The plates were incubated at 37°C with 5% CO2. After 14 days, the clone count was performed. Each experiment was performed in triplicate.

[0044] 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.

[0045] 4. Western Blot 5×10 5 A fresh CD34 + Cells were co-cultured for 10 days with phloroglucinol (20 μM) or DMSO (0.01% v / v) containing 10% serum, 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 lysed at 100°C for 10 min. 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.

[0046] 5. Reverse transcription-polymerase chain reaction (RT-PCR) The product uses Novizan HiScript Il Q RT SuperMix for qPCR (+gDNA wiper), and the installation steps are performed according to the instruction manual.

[0047] The primers used, HOXB4 primer sequence: Sequence (5'->3') F: TTGGAGCTGGAGAAGGAATT; Sequence (5'->3') R:CGAGCGGATCTTGGTGTTGG.

[0048] 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.

[0049] Example 1: Establishment of an in vitro circulating hematopoietic stem / progenitor cell (cHSPCs) culture system 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 + CD38 low / 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 low / 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 + CD38 low / 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.

[0050] 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).

[0051] 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.

[0052] Example 2: Treatment with the small molecule compound phloroglucinol significantly improved cHSPC amplification. This example investigated whether the small molecule compound phloroglucinol (purchased from MedChemExpress (MCE), lot number 108-73-6) could effectively promote cHSPC amplification.

[0053] The expression of TNCs and HSPC surface markers LIN, CD45RA, CD34, and CD38 was used as an efficiency indicator. Results are as follows: Figure 5 As shown, the addition of the small molecule compound phloroglucinol to the culture medium significantly increased LIN levels compared to the DMSO-treated control group. CD45RA CD34 + CD38 low / The number of HSPCs indicates that the small molecule compound phloroglucinol effectively promotes the amplification of cHSPCs.

[0054] Example 3: The small molecule compound phloroglucinol promotes cHSPC amplification by inhibiting inflammatory signaling from the microenvironment of NF-κB and JUN. Cultures were treated with the small molecule compound phloroglucinol to investigate whether phloroglucinol promotes cHSPC amplification by regulating NF-κB and JUN. Results are as follows: Figure 6 As shown, the data indicate that phloroglucinol inhibited the protein levels of total NF-κB p65 and its phosphorylated form (Ser536 site) and total c-Jun and its phosphorylated form (Ser63 site).

[0055] Example 4: The small molecule compound phloroglucinol promotes cHSPC amplification by promoting HOXB4 expression. The expression of the stem gene HOXB4 after treatment with the small molecule compound phloroglucinol was assessed by RT-PCR. Results are as follows: Figure 7 As shown, compared with the control group, phloroglucinol significantly promoted the expression of HOXB4.

[0056] Example 5: The potential of the small molecule compound phloroglucinol to promote cHSPC clonal formation. Through cloning experiments, such as Figure 8 As shown, compared with the control group, the small molecule compound phloroglucinol significantly increased the number of expanded hematopoietic stem and progenitor cells, indicating that phloroglucinol can enhance the clonogenic ability of expanded hematopoietic stem and progenitor cells.

[0057] 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 phloroglucinol 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 reconstruction.

2. The application according to claim 1, characterized in that, The working concentration of the phloroglucinol is 10~100μM.

3. 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.

4. A composition for in vitro expansion of human circulating hematopoietic stem / progenitor cells, characterized in that, Including phloroglucinol.

5. The composition according to claim 4, characterized in that, The final concentration of the phloroglucinol is 10~100 μM.

6. A culture medium for in vitro expansion of human circulating hematopoietic stem / progenitor cells, characterized in that, Includes the composition described in claim 4 or 5.

7. The culture medium according to claim 6, characterized in that, The culture medium also includes basal culture medium and hematopoietic support factors.

8. The culture medium according to claim 7, characterized in that, The hematopoietic support factors include SCF, TPO, FLT3L, IL-3, and IL-6.

9. A method for in vitro expansion of human circulating hematopoietic stem / progenitor cells for purposes other than disease diagnosis and treatment, characterized in that, This includes culturing peripheral blood mononuclear cells containing cHSPCs using the culture medium described in claim 7 or 8.

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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