Application of small-molecule inhibitor in maintaining resting state of human hematopoietic stem cell in-vitro culture functional characteristics to treat MLD

Abstract

The invention belongs to the technical field, and particularly relates to application of a small-molecule inhibitor in maintaining a resting state of human hematopoietic stem cell in-vitro culture functional characteristics to treat MLD. According to the invention, an efficient treatment scheme is provided for MLD, and through a hematopoietic stem cell transplantation gene therapy optimized by Bohemine, the ARSTA enzyme activity of a patient is remarkably improved and reaches the level of healthy people, and metabolic disorder and neuropathy are improved. Bohemine pretreatment can maintain the in-vitro resting state of the HSC, and ROS rise and self-renewal function loss caused by culture stress are avoided. The clone formation and transplantation reconstruction capability of the pretreated HSC is enhanced, the HSC is still close to fresh HSC after short-term culture, and the functional defect of in-vitro culture is solved. The therapy optimizes HSC culture conditions, improves the transgenosis integration rate and ARSA expression, enhances the treatment stability and success rate, has strong transformation potential, provides a basis for HSC culture optimization, promotes the application of HSC in cell and gene therapy, and has great significance.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to the application of a small molecule inhibitor in maintaining the functional characteristics of artificial hematopoietic stem cells in vitro and in a resting state for the treatment of MLD. Background Technology

[0002] Metachromatic leukodystrophy (MLD; OMIM: 250100) is a rare, inherited lysosomal storage disorder caused by a mutation in the ARSA gene that results in a deficiency in the function of the ARSA enzyme. Due to the lack of ARSA enzymes, sulfate metabolism is impaired, leading to the accumulation of these substances in the lysosomes of cells in the central and peripheral nervous systems. The continuous accumulation of sulfates in lysosomes leads to progressive demyelination and neurodegenerative changes. The clinical manifestation of this disease is developmental arrest, followed by a gradual loss of motor function, language ability, and cognitive abilities.

[0003] To date, various treatment methods have been used to treat metachromatic leukodystrophy (MLD), including allogeneic hematopoietic stem cell transplantation (allogeneic-HSCT), enzyme replacement therapy, umbilical cord transplantation, and lentivirus (LV)-based gene therapy (autologous-HSCT). Among these, bone marrow stem cell transplantation (HSCT) for MLD works on a phenomenon called "cross-correction," where healthy myeloid progenitor cells migrate to the brain and differentiate into microglia, secreting ARSA enzymes. These enzymes are absorbed by dysfunctional brain cells (such as oligodendrocytes), thereby improving sulfatate metabolism within neurons.

[0004] Interestingly, our research group previously developed a novel treatment method combining hematopoietic stem cell transplantation (HSCT) with gene therapy, using autologous hematopoietic stem cells (HSCs) transduced in vitro to carry LV vectors encoded by ARSA. After more than 12 years of IIT (investigator-initiated clinical trials), the use of LV-modified autologous HSCs successfully repaired neuropathological damage. Furthermore, as early as 2014, our group pioneered Asia's first hematopoietic stem cell gene therapy, applying it to symptomatic juvenile MLD patients and conducting a multicenter, single-arm, open-label clinical trial (ClinicalTrials.gov ID:NCT02559830). This trial assessed the long-term safety of the therapy by analyzing adverse events occurring during short-term and long-term follow-up after treatment, and evaluated clinical benefits using ARSA activity assays, MRI scores, and neurological function scores. Our research group, in collaboration with Guangzhou Women and Children's Medical Center, Shenzhen Second People's Hospital, Shenzhen Children's Hospital, and the University of Hong Kong, conducted a clinical study lasting nearly 10 years. The study showed that lentivirus-modified hematopoietic stem cell gene therapy (HSC-GT) is safe and effective for juvenile MLD patients after the onset of the disease. Patients with symptoms can still benefit from HSC-GT, bringing new hope for effective treatment of clinical patients. The results of this study were published in the journal Protein & Cell in 2024 (Lentivirus-modified hematopoietic stem cell gene therapy for advanced symptomatic juvenile metachromatic leukodystrophy: a long-term follow-up pilot study. Protein & Cell, 16(1), 16-27.).

[0005] Human hematopoietic stem cells (HSCs) are a very rare population of cells capable of producing all types of hematopoietic cells. In the human body, these cells are rich in CD34. + CD38 - / iow CD45RA - CD90 + Surface markers such as CD34 form specific cell populations. Most HSCs are in a quiescent state, possessing self-renewal capacity and multi-lineage differentiation potential. These characteristics make them powerful tools for regenerative medicine, cell and gene therapy. In the transplantation setting, CD34... +Cells are used as a heterogeneous cell product, containing hematopoietic progenitor cells, precursor cells, and HSCs—cells responsible for short-term and long-term blood cell production, respectively. The efficacy of HSC transplantation is closely related to the number of transplanted cells. HSCs from umbilical cord blood are increasingly used in transplantation therapy due to their ease of collection and lower immunogenicity compared to other allogeneic HSC sources. In gene therapy, CD34... + Cells typically need to be cultured for at least two days in a specific medium containing cytokines and growth factors (such as stem cell factor (SCF), thrombopoietin (TPO), and FLT3 ligand (FLT3L)) to promote HSC survival and facilitate gene modification via viral vectors. However, although growth factor signaling can support CD34... + While efficient gene correction in cells can occur, it can also lead to partial differentiation, resulting in the loss of HSC characteristics. Adult HSCs are known to exist in specific microenvironments that are hypoxic (i.e., hypoxic) and are involved in maintaining the functional properties of HSCs.

[0006] Furthermore, under hypoxic conditions, human CD34... + In vitro cell culture helps maintain the characteristics of HSCs. In fact, hypoxia can keep the reactive oxygen species (ROS) in HSCs at extremely low levels. These highly reactive molecules can induce oxidative stress and damage cells when levels are too high, but they can also act as signaling molecules, driving the differentiation and proliferation of HSCs.

[0007] Multiple cellular mechanisms can be used to scavenge reactive oxygen species (ROS) generated by physiological signals and various external stresses. Specifically, several enzymes work synergistically to limit the accumulation of highly reactive substances: the superoxide dismutase (SOD) family... - It is converted into H2O2, and then other enzymes from different families (such as thioredoxins, peroxidases, catalase, or glutathione peroxidase) can metabolize H2O2 to prevent its conversion into HO. ● These systems are crucial because elevated levels of reactive oxygen species (ROS) in HSCs can lead to their differentiation and loss of function, thereby reducing the body's ability to regenerate hematopoiesis. When CD34... + When cells (including HSCs) are used in gene therapy culture processes, they may partially lose function, primarily due to elevated reactive oxygen species levels caused by non-physiological O2 levels and growth factor signaling. Under these culture conditions, HSC function can be maintained through various methods.

[0008] Recent studies have proposed various HSC culture protocols, including culture under hypoxic conditions, in hydrophobic hydrogels, and in environments containing small molecules such as UM171.25-27. While these methods are highly attractive, hypoxic conditions and hydrogels are difficult to control in clinical applications, and their mechanisms of action remain unclear. Previous studies have found that antioxidant pretreatment can protect HSCs from reactive oxygen species damage induced by low-dose radiation. Based on this, it is hypothesized that in vitro antioxidant treatment of HSCs before gene therapy may help maintain their function.

[0009] Bohemine is a purine analogue and a synthetic selective CDK inhibitor with an IC50 of 1 / 3 for Cdk2 / cyclin E, Cdk2 / cyclin A, and Cdk9 / cyclin T1. 50 The half-maximal inhibitory concentrations (IC50) were 4.6 μM, 83 μM, and 2.7 μM, respectively. Bohemine also inhibited ERK2, with corresponding IC50 values ​​of 4.6 μM, 83 μM, and 2.7 μM. 50 The concentration is 52 μM, with relatively low inhibitory effects on CDK1, CDK4, and CDK6. To maintain the self-renewal function of human HSCs during in vitro culture, this invention uses Bohemine to protect HSCs from oxidative stress damage during in vitro culture, while also resisting the effects of radiation. Summary of the Invention

[0010] To overcome the shortcomings of the prior art, this invention demonstrates that Bohemine can protect HSCs from oxidative stress during culture, maintain their quiescent state in vitro, and preserve their stemness. It also confirms that in cell / gene therapy protocols, adding Bohemine to the culture medium helps improve the functional maintenance of HSCs in vitro.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the use of Bohemine in maintaining the resting state of the functional properties of human hematopoietic stem cells (HSCs) in vitro culture.

[0012] The second aspect of the present invention provides a method for maintaining the resting state of the functional characteristics of human hematopoietic stem cells in vitro, specifically: before long-term culture of human hematopoietic stem cells (HSCs), HSCs are first seeded into transduction medium and pre-cultured for 2 days with the addition of Bohemine; the transduction medium is prepared by adding 80-150 ng / mL stem cell factor (SCF), 90-130 ng / mL FMS-like tyrosine kinase 3 ligand (FLT3L), 50-70 ng / mL interleukin-3 (IL-3) and 7-15 nM thrombopoietin (TPO) to BIT medium.

[0013] Preferably, the working concentration of Bohemine is 200-500µM.

[0014] Preferably, StemSpan SFEM medium is used instead of BIT medium in the long-term culture of artificial hematopoietic stem cells.

[0015] Preferably, the artificial hematopoietic stem cells are derived from umbilical cord blood.

[0016] Although this study primarily focuses on hematopoietic stem cells (HSCs) in early postnatal cord blood samples, this method may also be applicable to HSCs at different developmental stages (more mature) in bone marrow or peripheral blood. Indeed, elevated reactive oxygen species (ROS) levels are commonly associated with HSC aging, and it is well known that adult HSCs are less efficient at hematopoietic remodeling than neonatal cord blood HSCs. Given the widespread use of bone marrow or mobilized peripheral blood CD34+ cells in autologous transplantation and gene therapy regimens, protecting these HSCs from further oxidative stress damage could help optimize gene therapy strategies. Overall, these results confirm that Bohemine promotes HSC maintenance and validate its effectiveness in protecting HSCs in vitro.

[0017] More preferably, monocytes in umbilical cord blood are separated using Ficoll gradient centrifugation, and then CD34 is purified by immunomagnetic separation using a CD34 microbead kit. + Cells, namely, artificial hematopoietic stem cells.

[0018] The third aspect of the present invention provides the use of artificial hematopoietic stem cells in the preparation of a medicament for treating metachromatic leukodystrophy (MLD), wherein the artificial hematopoietic stem cells are pre-cultured using the processing method described in the second aspect.

[0019] An ideal MLD treatment regimen must ensure targeted high expression of therapeutic ARSA transgenes, and this expression must simultaneously meet two conditions: maintaining the wild-type gene copy number and maintaining the undifferentiated quiescent state of HSCs in vitro culture, thereby avoiding unintended transgene integration and low ARSA enzyme activity. Bohemine-based HSC pretreatment technology provides a feasible approach for targeted gene high expression in specific cell types in the biomedical field. Multiple clinical and preclinical studies targeting hematopoietic stem and progenitor cells (HSPCs) and T cells have explored the application potential of this technology and successfully achieved targeted transgene integration at endogenous gene loci through the homology-directed repair (HDR) pathway. Since over 200 different ARSA mutations are known to cause benign MLD, an ideal treatment regimen must employ a universal gene correction strategy to achieve mutation-independent therapeutic effects.

[0020] To this end, the present invention employs a stepwise research method: first, hematopoietic stem cells (HSCs) are pretreated with Bohemine to maintain their stemness in vitro culture; then, HSCs are transfected, and sgRNAs with the highest editing efficiency (>87%) are screened out to evaluate the transgene integration efficiency (>40%) of endogenous ARSA sites in HSCs under unselected conditions. This process improves the transgene integration rate, thereby optimizing and enhancing the expression of ARSA enzymes, providing a feasible treatment method for MLD patients.

[0021] The difference in integration efficiency between green fluorescent protein (GFP) and ARSA transgenes highlights the importance of maintaining the quiescent state of HSCs in vitro. Studies have found that the ARSA integration frequency observed in patient-derived HSCs is low, which is significantly associated with long-term cryopreservation (>10 years) of HSCs and a decline in their quality compared to freshly isolated donor cells. This invention employs a carefully designed method of pretreating HSCs with Bohemine to enhance their functional properties in vitro and maintain their quiescent state, while simultaneously reducing ROS levels and oxidative stress. HSCs cultured with Bohemine exhibit superior hematopoietic reconstitution capacity in vivo, retaining long-term in vitro and in vivo functions. By inhibiting HSC cell differentiation, it maintains their in vitro function and quiescent state, delays mitochondrial activity, and thus optimizes and enhances ARSA enzyme expression, with expression levels comparable to those of differentiated myeloid progenitor cells from healthy adults.

[0022] In summary, this invention provides a feasible treatment for MLD patients, with proven efficacy and the potential for translation from laboratory to clinical practice. This proof-of-concept study further provides a mutation-independent treatment option for MLD patients, also with proven efficacy and translational potential from laboratory to clinical practice.

[0023] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0024] More preferably, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, antioxidants, adsorbents, filter aids, and release inhibitors.

[0025] Preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

[0026] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a breakthrough treatment for MLD. By using hematopoietic stem cell transplantation gene therapy optimized with the small molecule inhibitor Bohemine, patients' ARSA enzyme activity can be significantly restored to the level of healthy adults, effectively improving sulfatate metabolism abnormalities and neurodegenerative diseases.

[0027] In in vitro culture of HSCs, Bohemine can maintain their quiescent state, avoiding elevated ROS levels and loss of self-renewal function caused by culture stress. HSCs pretreated with Bohemine show significantly enhanced clonogenic and xenograft reconstitution capabilities, and retain characteristics close to those of fresh HSCs after short-term culture, thus solving the problem of stem cell functional defects caused by in vitro culture.

[0028] This therapy optimizes the in vitro culture conditions of HSCs, improves transgene integration rate and ARSA enzyme expression, and enhances treatment stability and success rate, demonstrating strong clinical translational potential. Simultaneously, it provides a scientific basis for optimizing HSC culture systems, promotes the application and development of hematopoietic stem cells in cell therapy and gene therapy, and is of great significance to MLD treatment and related research. Attached Figure Description

[0029] Figure 1 The diagram shows the correlation between experimental results and molecular mechanisms of hematopoietic stem cell (HSC) in vitro culture. A: Experimental design flowchart, B: LTC-IC experimental functional verification, C: Principal component analysis, D: Volcano plot, EF (heatmap + GSEA analysis), G: Epigenetic regulatory gene heatmap, H: Pathway GSEA analysis.

[0030] Figure 2Figure 1 shows the experimental results to verify the antioxidant and antioxidant stress protective effects of Bohemine on hematopoietic stem cells (HSCs); A: Flow cytometry results of direct ROS level measurement (left) and scatter plot (right); B: Antioxidant gene expression profile; C: Flow cytometry histogram (left) and bar chart (right).

[0031] Figure 3 Figure 1 shows the experimental results to verify the protective effect of Bohemine on hematopoietic stem cell (HSC) function; A: Primary CFU-C experiment, B: Secondary CFU-C experiment, C (5 weeks), D (10 weeks): "Long-term culture initiating cell (LTC-IC) frequency" was calculated using the limiting dilution method, E: "Human cell chimerism rate" was calculated, F: "Myeloid / lymphoid differentiation ratio" was analyzed, and G: "HSC phenotype cell ratio" was detected.

[0032] Figure 4 Figure 1 shows the experimental results illustrating the mechanism by which Bohemine maintains the in vitro function of hematopoietic stem cells (HSCs); A: HSC growth curve measurement; B: Analysis of immature surface markers (CD34). + CD90 + C: Differentiation rate observed through CFSE staining; DE: Cell cycle observation diagram; FG: Study of mitochondrial status.

[0033] Figure 5 The experimental results used to verify the effects of gene editing combined with Bohemine pretreatment on the gene correction efficiency and function of hematopoietic stem cells (HSCs) are shown in the figure below; A: Flow cytometry scatter plot, B: Quantification of eGFP at different MOIs. + C: Signal map of HDR (homology-directed repair) detected by ddPCR; DE: Quantification of HDR proportion in different treatment groups; F: Detection of ARSA mRNA by qPCR; G: ARSA enzyme activity; H: Myeloid differentiation analysis. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0036] Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the ARSA gene. This enzyme plays a crucial role in sulfatate metabolism in brain cells, and its deficiency leads to neurodegenerative diseases. This invention provides an allogeneic hematopoietic stem cell transplantation (allogeneic-HSCT) gene therapy (autologous-HSCT) that uses small-molecule inhibitors to maintain the functional properties of human hematopoietic stem cells in a quiescent state during in vitro culture. This therapy can serve as a potential treatment for MLD, and in clinical treatment, it significantly enhances the recovery of ARSA enzyme activity (>30-fold increase), reaching levels comparable to healthy adults. In summary, this research provides a proven and effective treatment for MLD patients, and this method has strong clinical translational potential.

[0037] In a resting state, the reactive oxygen species (ROS) level of hematopoietic stem cells (HSCs) is extremely low. Under stress, HSCs are activated and initiate proliferation and differentiation processes to ensure blood cell regeneration. Once activated, HSC ROS levels rise and act as signaling molecules to regulate these processes. However, after stress ends, HSC ROS levels must return to normal to prevent HSC depletion. Studies have shown that small molecule inhibitors can prevent the loss of HSC self-renewal function and maintain their quiescent state in vitro under various conditions. For example, fresh umbilical cord blood HSCs (DO CBHSCs) are mostly known to be in a quiescent state, characterized by slowed cell division and differentiation, delayed mitochondrial activation, and the preservation of their self-renewal potential without depletion. This phenomenon suggests that small molecule inhibitors can be used to maintain the self-renewal function of HSCs under culture-induced stress conditions, keeping them in a quiescent state.

[0038] HSCs are increasingly used in cell and gene therapy, which typically requires several days of in vitro culture. Studies have found that even short in vitro culture times can lead to severe defects in the self-renewal function of HSCs—their transcriptional program shifts from stem cell characteristics towards differentiation, making it impossible to maintain a quiescent state, consistent with expected outcomes. Furthermore, an interesting phenomenon was observed in HSC in vitro culture experiments: pretreatment of in vitro cultured HSCs with the small molecule inhibitor Bohemine significantly enhanced their clonogenic capacity in secondary clonogenic unit (CFU-C) assays and their remodeling capacity in xenograft models, both superior to untreated in vitro cultured HSCs. In summary, this invention demonstrates that adding Bohemine can protect the self-renewal function of HSCs during in vitro culture and maintain their quiescent state.

[0039] The results of this invention confirm that Bohemine can protect the function of HSCs in short-term (2-day) in vitro culture. Conventional two-day in vitro culture promotes the transformation of HSCs to progenitor cell characteristics. This culture process activates HSCs but also leads to a gradual loss of their self-renewal capacity and stem cell characteristics. Bohemine can enhance the generation of CFU-C, increase the frequency of long-term culture initiating cells (LTC-IC), and salvage the loss of in vivo hematopoietic reconstitution capacity caused by culture, making the characteristics of cultured HSCs closer to those of fresh day 0 HSCs (D0-HSCs).

[0040] This invention reveals that even short culture times can severely impair the self-renewal function of HSCs, specifically manifested as elevated ROS levels. Furthermore, the study confirmed that the small molecule inhibitor Bohemine can protect HSCs from culture-induced stress, maintain their self-renewal function, and keep them in a quiescent state in vitro. These findings lay the foundation for optimizing HSC culture conditions. By optimizing culture conditions, transgene integration can be improved, thereby enhancing ARSA enzyme expression and ultimately providing an effective treatment option for MLD patients.

[0041] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0042] 1. Experimental Methods 1.1 Umbilical cord blood samples and in vivo experiments Umbilical cord blood (CB) samples are collected from healthy infants in cooperation with the Guangdong Provincial Umbilical Cord Blood Hematopoietic Stem Cell Bank, and written informed consent from the mothers has been obtained before collection.

[0043] Hematopoietic stem cells (CD34) + The purification of CD34 cells followed a standard procedure: first, monocytes in the blood were separated using Ficoll gradient centrifugation, and then CD34 cells were purified using an immunomagnetic bead sorting method with a CD34 microbead kit (Miltenyi Biotech, Paris, France). + The cells are hematopoietic stem cells. Processed hematopoietic stem cells can be used directly in experiments, or cryopreserved in serum containing 10% DMSO (in liquid nitrogen) and used for subsequent experiments after thawing.

[0044] 1.2 Cell Culture During the co-incubation phase of Bohemine with hematopoietic stem cells, the complete culture medium used for cell transduction was prepared by adding 100 ng / mL stem cell factor (SCF), 100 ng / mL FMS-like tyrosine kinase 3 ligand (FLT3L), 60 ng / mL interleukin-3 (IL-3), and 10 nM thrombopoietin (TPO) (all purchased from PeproTech, USA) to BIT medium (STEMCELL Technologies). In subsequent long-term hematopoietic stem cell (HSC) culture experiments, StemSpan SFEM medium (STEMCELL Technologies) was used instead of BIT medium.

[0045] Drugs and treatment: The drugs used in the experiment and their concentrations are as follows: Bohemine (300µM), hydrogen peroxide (H2O2, 100µM), vascular endothelial growth factor A (VEGFA, 50ng / mL), ZM32388 (10nM), and Brivanib (50nM). Before starting long-term culture, hematopoietic stem cells needed to be pretreated with 300µM Bohemine, i.e., Bohemine was co-incubated with hematopoietic stem cells for 2 days.

[0046] The preparation method for 1 mM Bohemine stock solution is as follows: Dissolve 1 mg of Bohemine in 2.9375 mL of dimethyl sulfoxide (DMSO). The stock solution should be aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles; dilute to the working solution concentration before use.

[0047] 1.3 Flow Cytometry Analysis Cell surface staining was performed at room temperature in the dark. After staining, the cells were washed with PBS and resuspended. Hoescht and ZombiAqua (Biolegend) were used as viability markers for this staining.

[0048] Intracellular p38MAPK staining was performed using the phosphorus flow staining method (BD Biosciences), following the manufacturer's instructions: first, fix the cells with Fix Buffer I (BD Biosciences) at 37°C for 10 minutes, then permeabilize the cells on ice with Perm Buffer II (BD Biosciences) for 1 hour; finally, perform intracellular staining in PBS containing BSA and EDTA.

[0049] Cell analysis was typically performed on a BD Canto II or BD LSRII SORP flow cytometer, while cell sorting was performed on a BD Influx or BD Aria III SORP flow cytometer. All experimental results were analyzed using FlowJo software.

[0050] 1.4 Colony-forming unit (CFU-C) assay CFU-C culture began with 500 magnetically bead-sorted hematopoietic stem cells (HSCs) pre-cultured in Bohemine at 37°C in a CO2 incubator for 2 days (with no pretreatment as a control). Each experiment was performed in triplicate. The specific procedures were as follows: The HSCs were inoculated into H4435 methylcellulose medium (H4435, STEMCELL Technologies) and cultured for 12-14 days. Colonies were then identified and counted. Subsequently, culture plates with similar colony counts and types were resuspended in PBS buffer preheated to 25°C, and 1% of the recovered cells were inoculated into a secondary culture system.

[0051] 1.5 Long-term culture initiating cells (LTC-IC) assay After pre-culturing with Bohemine for 2 days (with no pretreatment as a control), hematopoietic stem cells (HSCs) were seeded using limiting dilution into 96-well plates pre-coated with MS5 stromal cells (using Myelocult medium, H5100, STEMCELL Technologies). Long-term colony counting-immunoculture assay (LTC-IC assay) was performed using Myelocult medium. The specific steps were as follows: cells were cultured continuously for 5 weeks, with half the volume of medium replaced weekly. After the culture was completed, the cells were harvested and seeded into 500 µL of methylcellulose medium (H4435). Colony growth was observed and evaluated after 10-12 days.

[0052] For extended long-term culture (LTC) experiments, hematopoietic stem / progenitor cells (HSPCs) were cultured in 6-well plates for 5 weeks, with half the culture medium volume replaced weekly; CD34 cells were sorted after the first 5 weeks of culture. + The cells (HSC cells) were then seeded into 96-well plates using limiting dilutions at different concentrations.

[0053] 1.6 Transplantation Experiment NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll) / SzJ(NSG) mice were housed in a pathogen-free animal facility. Adult NSG mice aged 8-12 weeks were selected and irradiated with a sublethal dose of 2.5 Gy using a GSRD1 irradiator, followed by isoflurane anesthesia before intravenous injection of human cells. All experimental procedures were strictly performed in accordance with animal ethics guidelines and met the requirements of the local ethics committee.

[0054] Hematopoietic stem cells (CD34) + Cells were pretreated (or not pretreated) and cultured in transduction medium for 2 days (the treatment group was treated with a final concentration of 300 µM Bohemine, while the control group was untreated). After cell counting, hematopoietic stem cells were intravenously injected into irradiated mice at a dose of 1–2.5 × 10⁻⁶ cells per mouse. 4 Each cell.

[0055] The mice were euthanized 16 weeks after transplantation, and four long bones were then removed for bone marrow analysis, and blood samples were collected for related tests.

[0056] 1.7 Microarray Transcription Analysis Transcriptome analysis was performed using the Affymetrix Human Clariom D chip, with three sorted hematopoietic stem cell (HSC) samples from different umbilical cord blood samples used for each experimental condition.

[0057] Data analysis was performed using TAC software, GSEA (gene set enrichment analysis), and molecular characterization software. The criteria for differentially expressed genes were: a fold change in gene expression ≥2 and a p-value ≤0.05. Heatmaps were generated using the ShinyHeatMap platform (http: / / shinyheatmap.com / ). All experimental data have been uploaded to the Array Express database (database ID: E-MTAB-12121).

[0058] 1.8 Measurement of ROS levels and mitochondrial activation ROS levels were measured on days 0 (D0), 1 (D1), and 2 (D2) after Bohemine treatment (i.e., co-incubation of Bohemine with hematopoietic stem cells for 2 days). The specific procedures are as follows: Cells were stained with surface antibodies, then incubated with CellRox DeepRED dye at 37°C for 30 minutes. After washing, they were fixed with BD cell fixation buffer and then analyzed on a BD Canto II flow cytometer. To verify the positive control with high ROS levels, some cells were incubated with TBHP for 30 minutes before being incubated with CellRox DeepRED probes.

[0059] After staining with surface antibodies, cells were incubated with CellRox Orange dye at 37°C for 30 minutes; after washing, they were incubated on ice and immediately analyzed by BD Canto II flow cytometry. If ROS induction is required, cells should be pretreated with Bohemine for 1 hour. ROS can be prepared by incubating with TBHP for 30 minutes before CellRox Orange staining, or by adding 100 µM H2O2 in the last 15 minutes of CellRoxOrange staining.

[0060] The procedure for measuring mitochondrial activation was as follows: After staining with surface antibodies, cells were mixed with 50 µM TMRE (tetramethylrhodamine ethyl ester) and 50 nM MTG (mitochondrial green fluorescent probe) in PBS, incubated at 37°C for 30 minutes, and then immediately analyzed on a BDCanto II or LSR-SORP flow cytometer.

[0061] 1.9 Expression of antioxidant genes or antioxidant profiles Antioxidant profiles were defined by detecting the relative expression levels of transcripts of 21 key antioxidant genes using real-time quantitative PCR (the detection method is described in the reference: Picou F, Vignon C, Debeissat C, et al. Bone marrow oxidative stress and specific antioxidant signatures in myelodysplastic syndromes. Blood Adv. 2019; 3(24):4271-4279. https: / / doi.org / 10.1182 / bloodadvances.2019000677.). The experiments were conducted using a Roche Applied Science LightCycler 480 microplate circulation system, combined with a universal probe library designed using probe discovery software for real-time quantitative PCR analysis. The relative expression levels of antioxidant genes were determined by 2... -ΔΔCt The method of calculation and analysis ultimately presents the overall outline of the antioxidant spectrum.

[0062] 1.10 Preparation of lentiviral vector ARSA LV The ARSA LV used in this study was prepared by transiently transfecting 293T cells with four plasmids (synthesized in vitro by GeneArt (ThermoFisher Scientific)). First, 293T cells were seeded in T162 culture flasks and amplified in Dulbecco's modified Eagle's medium (DMEM; Biochrom), then transferred to a cell factory in 10-well plates in Opti-MEM medium (ThermoFisher Scientific) for culture. Next, JetPEI transfection reagent (Polyplus transfection, ThermoFisher Scientific) was added to transfect the four plasmids: one encoding two core packaging constructs (pKLGag / pol and pKRev), one encoding a membrane construct (pK.G), and one encoding a transfer vector construct (pARSA). After incubating the transfected 293T cells in Opti-MEM medium for 24 hours, the 293T cell supernatant was collected and stored at 4°C. The culture medium for the 293T cells was then changed (the adherent 293T cells were first rinsed with DMEM medium, followed by the addition of DMEM medium supplemented with 10% fetal bovine serum (Gibco) and 1% L-glutamate (Biochrom),) and the cells were cultured for another 24 hours. A second collection of the 293T cell supernatant was then performed. The two supernatants were mixed and centrifuged at 280,000 × g for 4 hours at 4°C for viral concentration. The concentrated viral pellet was resuspended in Opti-MEM medium, mixed thoroughly, and then sterilized by filtration through a 0.2 μm filter and aseptically filled. Finally, the purified lentiviral vector was stored at -80°C.

[0063] 1.11 Flow Cytometry Analysis CD34 was enriched using the CliniMACS system (Miltenyi Biotec). + HSCs. In all isolated samples, the enriched HSCs from healthy donors and patients with post-bone marrow transplant lymphocytic dysplasia (MLD) had a purity >90%. 28 CD34 groups. + HSCs were cultured in StemMACS hematopoietic stem cell expansion medium (Miltenyi Biotec), which was supplemented with human stem cell factor (SCF; 100 ng / mL) and interleukin-3 (IL-3, 100 ng / mL; Miltenyi Biotec) for the first 3 days at 37°C and 5% CO2.

[0064] Subsequently, a two-stage myeloid differentiation protocol was employed. In the first stage (days 3-6), GM-CSF (50 ng / mL; Miltenyi Biotec) and M-CSF (50 ng / mL; Miltenyi Biotec) were additionally added to the culture medium. From days 6 to 9 (the second stage), only M-CSF (50 ng / mL) was added, and the maturation process of myeloid cells was finally monitored by flow cytometry. On day 10, cell analysis was performed using FITC-conjugated anti-CD33 (Miltenyi Biotec), PE-conjugated anti-CD14 (Miltenyi Biotec), PerCP-conjugated anti-CD45 (Miltenyi Biotec), APC-conjugated anti-CD11b (Miltenyi Biotec), and APC-conjugated anti-CD66b (Miltenyi Biotec) (BD FACSCalibur).

[0065] 1.12, qPCR and ddPCR Viral titers were determined using quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). To obtain viral DNA after LV capsid rupture, 2 mL of concentrated viral supernatant was digested with 2 IU DNase I (NEB, ThermoFisher Scientific), resulting in a final reaction volume of 40 μL. The sample was incubated at 37°C for 30 min, followed by treatment at 75°C for 15 min. Proteinase K (1 μL; Qiagen) was added to 5 μL of the DNase I digestion product for detection, bringing the final volume to 20 μL. The sample was then incubated at 50°C for 30 min and 98°C for 10 min, respectively.

[0066] qPCR was performed using a CFX real-time PCR instrument (Bio-Rad). The reaction system contained 2 μL of proteinase K digestion product, 12.5 μL of KAPA Probe FAST (Bio-Rad), 1 μL each of ITR primer (5 μM) and ITR Probe (5 μM), and 2.5 μL of water. The program was set as follows: 95℃ pre-denaturation for 3 minutes, followed by 40 cycles of 95℃ for 3 seconds and 60℃ for 20 seconds.

[0067] The ddPCR reaction system (Bio-Rad) was prepared as follows: The total volume of the ddPCR reaction system was 20 μL, and the specific components were as follows: ddPCR multiplex premix (13 μL), 950 nM primers (1 μL), 250 nM probe (1 μL), and 350 ng DNA template (5 μL). Simultaneously, ddPCR was performed on a C1000 Touch thermal cycler (Bio-Rad): 95℃ for 10 minutes, followed by 40 cycles of 95℃ for 30 seconds, 57℃ for 1 minute, and 72℃ for 2 minutes, with final enzyme inactivation at 98℃ for 10 minutes. Primer and probe information is detailed in Table 1.

[0068] Table 1. Oligonucleotide sequences used for qPCR and ddPCR Note: [1].Mern DS, Thome´ C. Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus forgene therapeutic approaches of intervertebral disc disorders. BMCMusculoskelet Disord 2015;16:341. DOI: 10.1186 / s12891-015-0799-4;[2].Lamsfus-Calle A, Daniel-Moreno A, Uren˜a-Baile´n G, et al. Universal genecorrection approaches for b-hemoglobinopathies using CRISPR-Cas9 and adeno-associated virus serotype 6 donor templates. CRISPR J 2021;4:207–222. DOI:10.1089 / crispr.2020.0141.

[0069] The specific experimental procedure is as follows: (1) DNA samples were divided into approximately 20,000 droplets using a QX200 droplet digital PCR (ddPCR) generator (Bio-Rad) and then transferred to 96-well plates; (2) Use the PX1 PCR plate heat sealer (Bio-Rad) to seal the 96-well plate to prevent liquid evaporation during the reaction; (3) Place the sealed 96-well plate on a C1000 Touch thermal cycler (Bio-Rad) for PCR reaction. The reaction program is as follows: initial denaturation at 95℃ for 10 minutes, followed by 40 cycles (95℃ for 30 seconds, 61℃ for 1 minute, 72℃ for 2 minutes), and finally end with enzyme inactivation at 98℃ for 10 minutes. (4) The reaction products were detected by a QX200 microdroplet analyzer, and the data were processed using QuantaSoft v1.6.6 analysis software (Bio-Rad).

[0070] 1.13, qRT-PCR On day 10 post-HSC transfection, total RNA was extracted using the RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR was performed using the CFX96™ real-time quantitative PCR system (Bio-Rad), with cDNA amplification and quantification completed using the KAPA SYBR FAST 2×MasterMix (KAPABiosystems).

[0071] The experimental results were standardized using the expression level of β2 microglobulin (β2M) as an internal reference. The crossover point (CP) value of unknown samples was determined using Formula 2 (CP). β2M -CP target gene ) Calculation. The qRT-PCR primers used in this experiment are specific (primer source reference: Johnson RL, Milenkovic L, Scott MP. In vivo functions of thepatched protein: requirement of the C terminus for target gene inactivation but not hedgehog sequestration. Mol Cell 2000;6:467–478. DOI: https: / / doi.org / 10.1016 / S1097-2765(00)00045-9.), detecting only ARSA transcripts (transgene specific) and not cross-reacting with the expression of endogenous ARSA.

[0072] For samples that did not undergo transgenic transduction (i.e., the control group), no signal was detected in the qRT-PCR analysis. Therefore, their Ct values ​​were set to the maximum number of cycles (40 in this experiment) to calculate fold change. This approach is consistent with the methods used in several analysis software programs (such as Applied Biosystems DataAssist v3.0 and Integromics RealTime StatMiner), which assign the maximum Ct value to undetected values. It should be noted that this method may lead to a bias in the actual fold change (i.e., overestimation), but it can be used to plot the relative expression level of mRNA in Bohemine + lentivirus (LV) treated samples relative to the control group.

[0073] 1.14. ARSA enzyme activity detection The quantitative detection of ARSA (arylsulfatase A) enzyme activity was performed using an artificial substrate, p-nitrocatechol sulfate (pNCS; Merck). The specific method was described in the manufacturer's instructions, and the steps are as follows: (1) Sample preparation: Prepare cell lysis buffer with a concentration of 2.5 × 10⁻⁶. 6 Cells / mL

[0074] (2) Reaction system: The cell lysate was mixed with the substrate solution, which consisted of 10 mM pNCS, 0.5 mM sodium pyrophosphate, and 10% NaCl, dissolved in 0.5 M sodium acetate buffer (pH 5.0).

[0075] (3) Incubation reaction: The mixed reaction system was incubated at 8°C for 48 hours.

[0076] (4) Termination and detection: After the reaction is completed, 0.5M NaOH is added to terminate the reaction. The absorbance value is measured at a wavelength of 514nm by spectrophotometry to reflect the conversion rate of the substrate and thus quantify the ARSA enzyme activity.

[0077] This method directly reflects the functional activity of ARSA enzymes by detecting the conversion efficiency of artificial substrates, and is a key experimental tool for evaluating the biological function of this enzyme.

[0078] 1.15 Statistical Analysis Data are expressed as mean ± standard deviation. Statistical significance was determined by t-test or Fisher's exact test between two groups and by one-way ANOVA between three groups. , , ,and The results were considered statistically significant. Statistical analysis was performed using GraphPad Prism version 6.01. In vivo and in vitro limiting dilution analyses were performed using L-Calc software.

[0079] 2. Experimental Results 2.1 Changes in HSC characteristics and HSC gene expression function caused by in vitro culture To understand the impact of in vitro culture on the functional properties of HSCs, a limited-dilution LTC-IC experiment (an experimental model for assessing immature HSC function) was conducted. The results are as follows: Figure 1 As shown, A is the experimental design flowchart, illustrating the research framework from HSC sorting to grouping "uncultured (D0-HSC)" and "cultured for 2 days (D2-HSC)", and then to functional experiments (LTC-IC) and molecular experiments (microarray); B is the functional validation of the LTC-IC experiment, which uses the limiting dilution method to count the "cell frequency of colony generation" and quantifies the difference in stem cell activity between D0 / D2-HSC (D2 has lower activity); C is principal component analysis, showing the clustering and separation of D0 / D2-HSC, indicating that the gene expression profiles of the two groups are "overall different"; D is a volcano plot, screening for differentially expressed genes, with red and blue dots representing significant up- and down-regulation. Genes: Quantifying the scale of gene expression changes caused by "2 days of culture" (5476 differentially expressed genes); EF (heatmap + GSEA analysis) focuses on "stem cell characteristic-related gene sets", with heatmaps visualizing the expression differences of HSC / progenitor cell characteristic genes, and GSEA further statistically analyzing "stem gene sets enriched in D0 and differentiation gene sets enriched in D2"; G is a heatmap of epigenetic regulatory genes, showing the impact of culture on key regulatory factors of HSC stemness / differentiation; H is a pathway GSEA analysis, with D0 enriching stemness maintenance pathways such as "hypoxia and tumor suppression (P53)" and D2 enriching differentiation / metabolic pathways such as "cell cycle and oxidative phosphorylation".

[0080] First, according to CD34 + CD38 - CD45RA - CD90 + Phenotyping was performed by purifying human HSCs through cell sorting. The cells were then seeded directly under LTC-IC conditions (D0-HSCs (uncultured HSCs)) without culture, or D2-HSCs (HSCs cultured on day 2) were seeded after 2 days of culture. Figure 1A) Inoculated under LTC-IC conditions. Subsequently, the colony-forming cell frequencies of D2-HSCs and uncultured HSCs (D0-HSCs) were compared under LTC-IC conditions. Under LTC-IC conditions, the frequency of D2-HSCs was (1 / 9, 95% CI [1 / 11–1 / 8]), while the frequency of D0-HSCs was (1 / 4, 95% CI [1 / 5–1 / 4]). The colony-forming cell frequency of D2-HSCs after 5 weeks was 2.25 times lower than that of D0-HSCs, indicating that D2-HSCs lost their HSC stem cell characteristics during culture. Figure 1 (B in the original text). To further understand the molecular mechanisms of these functional changes, transcriptomic analysis was performed using CLARIOM D microarray technology, and the transcriptomic profiles between D2-HSCs and D0-HSCs were compared. Principal component analysis showed that D2-HSCs and D0-HSCs clustered separately (B in the original text). Figure 1 The C in the D2-HSC revealed different transcriptome profiles. A total of 5476 genes were differentially regulated under both conditions, with 1868 genes downregulated and 3608 genes upregulated in the D2-HSC. Figure 1 (D in the original text). First, gene set enrichment analysis (GSEA) was performed using certain C2-CGPHSC and progenitor-specific gene sets from https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp. Figure 1 The EF in D2-HSCs was found to be enriched in D0-HSCs. Compared to D0-HSCs, HSC and progenitor gene signatures were downregulated and upregulated in D2-HSCs, respectively, confirming that a 2-day in vitro culture period induced HSC differentiation and led to HSC function loss. Figure 1 In addition, some genes involved in epigenetic modification, such as TET2, DNMT3B, EZH1, and EZH2, were found to have different expression levels when comparing D2-HSC and D0-HSC conditions. Specifically, EZH2 expression was increased in D2-HSC, which is related to HSC activation and differentiation; while EZH1 expression was decreased, which is related to the stemness characteristics of HSC. Similarly, TET2 expression was also lower in D2-HSC than in D0-HSC. Figure 1 (G in the text). Finally, this analysis showed that in HSCs, culture-induced cell cycle / differentiation programs and oxidative stress persisted for 2 days. When gene ontology analysis was performed using GSEA software, the signature pathways enriched after 2 days of culture were associated with cell cycle and oxidative phosphorylation. Conversely, the features enriched in DO-HSCs were associated with hypoxia (G in the text). Figure 1(H in H). In fact, it has been reported that the molecular characteristics exhibited by HSCs are associated with hypoxia regardless of oxygen concentration. Therefore, in vitro culture not only drives changes in the functionality of HSCs but also triggers metabolic shifts.

[0081] 2.2 Bohemine reduces ROS and oxidative stress. To verify whether Bohemine could maintain the fertility of hematopoietic stem cells (HSCs) during a two-day stress culture period, the antioxidant effect of Bohemine was first examined in HSCs. The experimental procedure was as follows: HSCs were pretreated with Bohemine before oxidative stress induction, followed by oxidative stress induced by tert-butyl hydroperoxide (TBHP), and reactive oxygen species (ROS) levels were detected using the CellRox orange probe. The results showed that Bohemine could inhibit the TBHP-mediated increase in ROS levels in human HSCs, indicating its antioxidant activity. Therefore, ROS levels in Bohemine-treated and untreated HSCs were further measured on day 0 (uncultured) and day 2 (D2). The experimental results are as follows: Figure 2 As shown, A represents direct measurement of ROS levels, while the left side shows flow cytometry results using the CellROX deep red probe, comparing the "Bohemine-treated group (light purple peak)" with the "untreated D2-HSC" group. The fluorescence intensity (MFI) of the "group (gray peak)" visually demonstrates that Bohemine reduces ROS, and the scatter plot on the right quantifies the "multiplier increase in ROS level," further verifying the inhibitory effect of Bohemine on ROS. B uses "antioxidant gene expression profile" to indirectly corroborate this, comparing the antioxidant gene expression of D0-HSC (dashed line, representing the baseline without oxidative stress), D2-HSC (thick black line, representing the state under oxidative stress), and Bohemine-treated D2-HSC (blue line). The curve after Bohemine treatment approaches D0, indicating that it alleviates oxidative stress by regulating antioxidant genes. C's left-hand flow cytometry histogram compares the phosphorylated p38MAPK levels of the Bohemine-treated group (light purple peak) and the untreated D2-HSC group (gray peak); the right-hand bar chart quantifies the difference, proving that Bohemine reduces p38MAPK phosphorylation, indirectly indicating that it reduces ROS generation and resists oxidative stress.

[0082] The results showed that, under untreated conditions, the ROS levels of HSCs increased from D0 to D2. Furthermore, when cells were treated with Bohemine, the ROS levels tended to decrease. Figure 2(A in the original text). To reinforce these results, the antioxidant effect of Bohemine on HSCs was assessed using the "Antioxidant Map" assay after 2 days of culture. The "Antioxidant Map" assay provides a comprehensive view of the activation of multiple antioxidant pathways in response to increased intracellular ROS levels by tracking the expression levels of key antioxidant gene transcripts. Compared to uncultured cells (D0-HSCs), many antioxidant genes were significantly overexpressed in D2-HSCs, confirming that D2-HSCs were indeed affected by oxidative stress compared to D0-HSCs. Figure 2 In the figure, B, D2-HSC, and D2-HSC are shown as thick black lines, while D0-HSC is shown as a dashed line; the difference can be seen by comparing the two. Conversely, the expression curve of antioxidant genes in D2-HSC after Bohemine treatment ( Figure 2 The B line (Bohemine D2-HSC, blue line) shifted towards D0-HSC, indicating that many analyzed genes were downregulated in the presence of Bohemine. To further demonstrate the role of Bohemine in inhibiting ROS generation in culture, the phosphorylation status of p38MAPK (p38 mitogen-activated protein kinase), a secondary messenger of oxidative stress, was investigated. After two days of culture, compared with untreated D2-HSC, the phosphorylation of p38MAPK in Bohemine-pretreated D2-HSC was significantly reduced, but still present (…). Figure 2 The presence of C in the figure indicates that the ROS level decreased after Bohemine treatment. These results suggest that Bohemine can reduce the ROS level in D2-HSCs, and that Bohemine pretreatment can protect HSCs from oxidative stress during a 2-day culture period.

[0083] 2.3 Bohemine-treated HSCs exhibit better hematopoietic reconstitution in vivo, preserving the long-term in vitro and in vivo functions of HSCs. The effects of Bohemine treatment on HSC function were investigated; CFU-C experiments were performed using D2-HSCs, and their immature functions were further explored through their reculturing ability (serial CFU-C assays). In short, HSCs, with or without Bohemine treatment, were sorted, cultured in complete medium for 2 days, and then inoculated into CFU-C medium. The experimental results are as follows: Figure 3As shown, A is the "primary CFU-C experiment," demonstrating that Bohemine does not affect the initial colony formation of HSCs; B is the "secondary CFU-C experiment," showing that the Bohemine-treated group has more colonies, indicating that it can maintain the HSC colony formation potential and prevent culture-induced functional loss. C (5 weeks) and D (10 weeks) used the limiting dilution method to count the "long-term culture initiating cell (LTC-IC) frequency." The Bohemine-treated group had a higher LTC-IC frequency (close to D0-HSCs), proving that it can protect the immature function of HSCs, and the effect can be maintained even with prolonged culture; E counted the "human cell chimerism rate," and the Bohemine-treated group had a higher chimerism rate and lower heterogeneity, indicating that it improves the in vivo reconstruction efficiency of D2-HSCs; F analyzed the "myeloid / lymphoid differentiation ratio," showing no significant deviation, proving that Bohemine does not affect the HSC differentiation direction; G detected the "HSC phenotypic cell ratio," and the treated group showed an increasing trend, indirectly supporting its protection of HSC function.

[0084] The results showed that the number of colonies formed under both conditions was comparable in primary culture. Figure 3 In secondary CFU-C culture, Bohemine-pretreated D2-HSCs showed a significantly increased colony count compared to untreated D2-HSCs (A in the text). Figure 3 (B) For two independent cord blood samples, the clonogenic capacity of HSCs was measured, comparing D2-HSCs (untreated D2-HSCs and Bohemine-treated D2-HSCs) with uncultured D0-HSCs. The secondary clonogenic capacity of untreated D2-HSCs was decreased compared to uncultured D0-HSCs, while Bohemine-pretreated D2-HSCs prevented this functional loss. In one experiment, tertiary CFU-C assays were performed, revealing that Bohemine-pretreated D2-HSCs generated more clones. This suggests either better preservation of immature progenitor cells or enhanced HSC self-renewal capacity, resulting in a greater number of CFU clones formed during secondary (and tertiary) refolding in the case of Bohemine pretreatment. Furthermore, no bias in CFU-C type was observed, demonstrating that Bohemine did not affect HSC differentiation. To further confirm the effect of Bohemine on HSCs, the potential impact of Bohemine on the self-renewal potential of D2-HSCs was assessed. The frequency of LTC-IC (long-term culture initiation cells, i.e., the least mature cells) was quantified using a limiting dilution assay. The results showed that Bohemine pretreatment promoted the maintenance of LTC-IC in D2-HSCs compared to untreated D2-HSCs, with the frequency of LTC-IC approaching that of D0-HSCs (1 / 5, 95% CI [1 / 6–1 / 5]). Figure 3(C in the text). To assess the function of Bohemine in maintaining more immature HSCs, similar experiments were performed, including an extended (10-week) culture period. After the first 5 weeks of batch culture, persistent CD34 in LTCs were obtained by sorting. ⁺ Cells were seeded using a limiting dilution method for 5 weeks, after which their colony-forming ability was assessed. After 10 weeks of culture, untreated D2-HSCs (1 / 98, 95% CI [1 / 81–1 / 118]) showed the best colony-forming ability. Figure 3 Compared to D), the LTC-IC frequency in D2-HSC treated with Bohemine was 50% higher (1 / 66, 95% CI [1 / 55-1 / 79]), which suggests that Bohemine has the potential to protect the immature functions of HSC.

[0085] To verify these results, CD34 + Cells were pretreated with Bohemine or untreated and cultured in complete medium for 2 days (D2-CD34). + Cells were then injected into immunodeficient NSG mice (NOD-PrkdcscidIl2rgem1 / Smoc). Sixteen weeks post-transplantation, the mice were sacrificed, and the human chimera was assessed using a specific anti-human CD45 antibody to evaluate its long-term reconstitution potential. Results showed that D2-CD34... + The cells generated human hematopoietic progenitor cells in the body, but their efficiency was not as high as that of fresh CD34 cells. + Cells (D0-CD34) + ), because D2-CD34 was transplanted into the bone marrow of NSG mice. + hCD45 was detected in the cells. + High heterogeneity at the cellular level Figure 3 In E), this heterogeneity is observed in D2-CD34 pretreated with Bohemine. + The condition was reduced. In fact, D2-CD34 pretreated with Bohemine was injected with [a substance that was] reduced. + In mice injected with untreated D2-CD34 cells, 86% showed human chimerism, a proportion >10%, while... + In mice with this cell line, the proportion was 57% ( Figure 3 In E). Neither myeloid cell bias nor lymphoid cell bias was detected. Figure 3 (F in the text). However, using the classic HSC marker, in D2-CD34 pretreated with Bohemine... + A trend toward higher HSC phenotype levels was observed in mouse bone marrow cells (although not significantly); Figure 3(G in the text). In addition, a sequential transplantation experiment was conducted. In primary recipient mice, there was no difference in the percentage of chimerism between the two conditions, but in secondary recipients, Bohemine-pretreated D2-CD34... + The cells were more efficient, indicating that HSCs maintained their function better when treated with Bohemine during culture. In summary, these data suggest that Bohemine may preserve the hematopoietic remodeling function of HSCs during a 2-day culture period. Figure 3 (G in the middle).

[0086] 2.4. Bohemine maintains the in vitro function of HSCs by restricting HSC differentiation, ensuring the in vitro resting state of HSCs and delaying mitochondrial activity. To understand how Bohemine maintains the in vitro function of HSCs, several parameters were monitored, including proliferation, expression of immature cell surface markers, and cell division rate at different culture time points. The experimental results are as follows: Figure 4 As shown, A is the HSC growth curve, proving that Bohemine has no significant effect on "cell number growth"; B is the analysis of "immature surface markers (CD34)". + CD90 + The marker was lost with prolonged culture time, but there was no difference between the Bohemine-treated and untreated groups. C, using CFSE staining to observe differentiation rate, showed a lower differentiation rate in the Bohemine-treated group, proving that it could "delay HSC differentiation." DE, observing the cell cycle, showed that more HSCs in the Bohemine-treated group were in the quiescent phase (G0 phase). FG investigated mitochondrial status; F showed no significant difference in mitochondrial quality; G, using the TMRE probe, found a lower mitochondrial membrane potential (delayed activation) in the Bohemine-treated group, indicating that it has a protective function for HSCs.

[0087] The results showed that Bohemine had no significant effect on HSC cell growth. Figure 4 (A) As expected, with prolonged culture time, the HSC phenotype (CD34) + CD90 + The D2-CD34 was gradually lost, and no D2-CD34 was observed in either untreated or Bohemine-pretreated samples. + Differences between cells ( Figure 4 (B in the text). However, compared with untreated D2-HSCs, the differentiation rate of HSCs pretreated with Bohemine (CFSE (carboxyfluorescein diacetate succinimide) staining) was decreased (B in the text). Figure 4(C in the text). Therefore, the cell cycle status of HSCs was examined after two days of culture. Bohemine-pretreated D2-HSCs were clearly more in a quiescent state, which explains the delay observed in the CFSE cell differentiation assay. Figure 4 The mitochondrial membrane potential was measured using the TMRE probe, and mitochondrial mass was detected using the MTG probe. The mitochondrial activation in D2-HSCs pretreated with Bohemine and untreated D2-HSCs was analyzed. It was observed that although Bohemine had a very limited effect on mitochondrial mass (D-4E). Figure 4 In the F group, mitochondrial activation was delayed after culturing HSCs pretreated with Bohemine for 2, 4, and 7 days. Figure 4 (G in the text). In summary, this invention demonstrates that Bohemine protects the maintenance of HSC function in vitro by limiting cell differentiation rate and metabolic activation.

[0088] 2.5 Bohemine pretreatment of HSCs for the treatment of metachromatic leukodystrophy (MLD) Lentiviral vector ARSA LV was transfected into healthy donor-derived hematopoietic stem cells (HSCs) in DMEM medium with different multiplicity of infection (MOIs) of 2000, 1000, and 500 for 14 hours. Immediately after gene editing, the HSCs were cultured in myeloid differentiation medium (StemMACS) for 10 hours. Ten days after gene editing and lentiviral transduction, the gene correction efficiency of HSCs was assessed using various molecular analyses, including ddPCR, qPCR, and ARSA enzyme activity assays. Simultaneously, flow cytometry analysis was performed on cells transduced with eGFP (GenBank: NG_009260.2) and ARSA (GenBank: U55762.1) donor templates. It is important to note that eGFP expression was only induced after successful integration into the endogenous ARSA locus, a process regulated by the endogenous ARSA promoter. Experimental results are as follows: Figure 5 As shown, A is a flow cytometry scatter plot that visually displays the eGFP of the "Bohemine+LV group". + The proportion of cells was much higher than that of the control group; B represents the quantification of eGFP at different MOIs. +The proportions show that the integration rate exceeded 47% at MOI=1000, verifying the effectiveness of the strategy; C is the signal map of HDR (homology-directed repair) detected by ddPCR, and DE is the HDR proportion of different treatment groups, which corresponds to the flow cytometry results, proving that the integration rate of the Bohemine pretreatment group is higher; F is the "ARSA mRNA" detected by qPCR, and the expression level of the Bohemine treatment group is 220 times higher than that of the control group, proving that the integrated gene can be effectively transcribed; G is ARSA enzyme activity, indicating that gene editing / Bohemine treatment does not destroy the original function of HSCs; H is myeloid differentiation analysis, analyzing myeloid markers such as CD33 / CD11b / CD66b, and the proportions of each group are similar, proving that Bohemine+LV treatment does not affect the differentiation direction of HSCs.

[0089] Notably, the results showed that the strategy of this invention could integrate eGFP into more than 47% of transduced cells (HSCs) at a multiplicity of infection (MOI) of 1000. Figure 5 (AB in the text). Other MOI values ​​(500 and 2000) achieved eGFP transgene integration rates of 37% and 39%, respectively. Analysis of the same batch of samples by droplet digital PCR (ddPCR) validated the results obtained by flow cytometry (FACS): HSCs pretreated with Bohemine and cultured in vitro showed a 40.6% transgene integration rate after LV-eGFP (MOI 1000) transduction; while HSCs without Bohemine pretreatment and HSCs treated only with LV showed no positive signal. Figure 5 In Bohemine-pretreated HSCs transduced with ARSA cDNA using LV vectors, a multiplicity of infection (MOI) of 2000 showed the best integration efficiency (32.7%). Figure 5 The integration rate of ARSA cDNA was significantly lower in the group with an E1 of LV, while that of the group with an MOI of 500 was 25.8% (p < 0.01). To assess the endogenous transcription of integrated ARSA cDNA, qPCR primers and probes were designed that bound only to ARSA mRNA in the Bohemine-pretreated group and not to endogenous wild-type ARSA mRNA. In this qPCR experiment, it was observed that the expression level of ARSA mRNA in Bohemine-treated HSCs was more than 220-fold higher than that in cells treated with LV alone or in untreated samples from healthy donors. Figure 5 The generated data confirmed that the designed probe did not hybridize with classical ARSA mRNA. Furthermore, because these cells were derived from healthy donors, their ARSA enzyme activity remained unchanged regardless of whether they underwent gene editing and correction. Figure 5(G in the text). Additionally, to test whether Bohemine+LV vector treatment affected HSC cell differentiation, specific myeloid markers were analyzed. Results showed that regardless of the treatment method, the proportions of cells expressing CD33 (range 98.6-99.5%), CD11b (37.0-39.7%), or CD66b (23.0-28.1%) were similar. Figure 5 The presence of H indicates that Bohemine+LV vector treatment can improve transgene integration efficiency, thereby maintaining HSC cell differentiation.

[0090] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of Bohemine in maintaining the resting state of functional characteristics of human hematopoietic stem cells in vitro.

2. A method for maintaining the resting state of the functional characteristics of artificial hematopoietic stem cells in vitro, characterized in that, Before long-term culture of artificial hematopoietic stem cells, the artificial hematopoietic stem cells are first seeded into transduction medium and pre-cultured for 2 days with the addition of Bohemine. The transduction medium is prepared by adding 80-150 ng / mL stem cell factor, 90-130 ng / mL FMS-like tyrosine kinase 3 ligand, 50-70 ng / mL interleukin-3 and 7-15 nM thrombopoietin to BIT medium.

3. The method for maintaining the resting state of the functional characteristics of artificial hematopoietic stem cells in vitro according to claim 2, characterized in that, The working concentration of Bohemine is 200-500 µM.

4. The method for maintaining the resting state of the functional characteristics of artificial hematopoietic stem cells in vitro according to claim 2, characterized in that, In the long-term culture of artificial hematopoietic stem cells, StemSpan SFEM medium was used instead of BIT medium.

5. The method for maintaining the resting state of the functional characteristics of artificial hematopoietic stem cells in vitro according to claim 2, characterized in that, The artificial hematopoietic stem cells are derived from umbilical cord blood.

6. The method for maintaining the resting state of the functional characteristics of artificial hematopoietic stem cells in vitro according to claim 5, characterized in that, Mononuclear cells were isolated from umbilical cord blood using Ficoll gradient centrifugation, and CD34 was then purified by immunomagnetic separation using a CD34 microbead kit. + Cells, namely, artificial hematopoietic stem cells.

7. The application of artificial hematopoietic stem cells in the preparation of drugs for treating metachromatic leukodystrophy, characterized in that, The artificial hematopoietic stem cells are pre-cultured using the processing method described in any one of claims 2-6.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

10. The application according to claim 8, characterized in that, The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

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