CN120944819B

CN120944819B - Application of small molecule inhibitor in treating MLD (tumor necrosis factor) by maintaining in-vitro culture functional characteristics of human hematopoietic stem cells

Info

Publication number: CN120944819B
Application number: CN202511475680.7A
Authority: CN
Inventors: 刘佳; 连祺周; 萧亮; 曲寿康
Original assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Current assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Priority date: 2025-10-16
Filing date: 2025-10-16
Publication date: 2025-12-23
Anticipated expiration: 2045-10-16
Also published as: CN120944819A

Abstract

The invention belongs to the technical field, and particularly relates to application of a small molecule inhibitor in treating MLD (tumor necrosis factor) by maintaining in-vitro culture functional characteristics of human hematopoietic stem cells. The invention provides a high-efficiency treatment scheme for MLD, and through Bohemine optimized hematopoietic stem cell transplantation gene therapy, the ARSA enzyme activity of a patient is obviously improved, the level of a healthy person is reached, and metabolic abnormality and neuropathy are improved. Bohemine pretreatment can maintain HSC in vitro resting state, and avoid ROS increase and self-renewal function loss caused by culture stress. The pre-treated HSC clone formation and transplantation reconstruction capability are enhanced, short-term culture is still close to fresh HSC, and the functional defect of in vitro culture is overcome. The method optimizes the culture condition of the HSC, improves the integration rate of the transgene and the ARSA expression, enhances the treatment stability and the success rate, has strong transformation potential, provides basis for the culture optimization of the HSC, and promotes the application of the HSC in cell and gene therapy, thereby having great significance.

Description

Application of small molecule inhibitor in treating MLD (tumor necrosis factor) by maintaining in-vitro culture functional characteristics of human hematopoietic stem cells

Technical Field

The invention belongs to the technical field, and particularly relates to application of a small molecule inhibitor in treating MLD (tumor necrosis factor) by maintaining in-vitro culture functional characteristics of human hematopoietic stem cells.

Background

Metachromatic leukodystrophy (MLD; OMIM: 250100) is a rare inherited lysosomal storage disorder, resulting from mutations in the ARSA gene that lead to defective ARSA enzyme function. Due to the deficiency of the ARSA enzyme, the metabolism of sulfate esters is impaired, resulting in accumulation of these substances in the lysosomes of cells of the central and peripheral nervous system. The constant accumulation of sulfate in lysosomes can lead to progressive demyelination and neurodegeneration. The clinical manifestation of the disease is developmental arrest followed by progressive loss of motor function, language ability and cognitive ability.

To date, a variety of therapeutic approaches have been used for the treatment of 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 them, the principle of bone marrow stem cell transplantation (HSCT) for MLD is based on a phenomenon known as "cross correction", i.e., migration of healthy myeloid precursor cells to the brain and differentiation into microglia, secretion of ARSA enzymes, which are taken up by brain cells lacking functions (e.g., oligodendrocytes), thereby improving sulfate metabolism in neurons.

Interestingly, a new therapeutic approach was previously developed in this group of subjects, combining Hematopoietic Stem Cell Transplantation (HSCT) with gene therapy, carrying an ARSA-encoded LV vector by in vitro transduction of autologous Hematopoietic Stem Cells (HSCs). Clinical studies with 12+ years IIT (clinical trial initiated by researchers) showed that the use of LV modified autologous HSCs successfully repaired neuropathic lesions. Meanwhile, the subject group has initiated asian first hematopoietic stem cell gene therapy as early as 2014, applied it to symptomatic adolescent MLD patients, and developed a multi-center, single arm, open clinical trial (clinical three.gov ID: NCT 02559830) to evaluate the long-term safety of the therapy by analyzing adverse events occurring during short-term and long-term follow-up after treatment, and evaluate clinical benefit by ARSA activity detection, MRI score, and neurological score, etc. The subject group combines medical institutions such as Guangzhou female children medical center, shenzhen second people hospital, shenzhen children hospital and hong Kong university to develop clinical researches for nearly 10 years, and the research shows that the slow virus modified hematopoietic stem Cell gene therapy (HSC-GT) is safe and effective for the sick teenager MLD patients, the symptomatic patients can still benefit from the HSC-GT, a new hope is brought for the effective treatment of clinical patient groups, and the research results are published in Protein & Cell journal 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.）.

Human Hematopoietic Stem Cells (HSCs) are a very rare population of cells with the ability to produce all hematopoietic cells. In humans, such cells are enriched for surface markers such as CD34 ⁺、CD38^-/iow、CD45RA^-、CD90⁺, which constitute a specific cell population. HSCs are mostly in a resting state, with self-renewal capacity and multipotent differentiation potential. These properties make it a powerful tool for regenerative medicine, cell and gene therapy. In the context of transplantation, CD34 ⁺ cells are used as a heterogeneous cell product comprising hematopoietic progenitor cells, precursor cells and HSCs, which are responsible for short-term and long-term hematopoiesis, respectively. The effect of HSC transplantation is closely related to the number of transplanted cells. HSCs in cord blood are increasingly being used in transplantation therapy because of their ease of collection and lower immunogenicity compared to other allogeneic sources of HSCs. In gene therapy, CD34 ⁺ cells are typically cultured in a specific medium containing cytokines and growth factors, such as Stem Cell Factor (SCF), thrombopoietin (TPO), and FLT3 ligand (FLT 3L), for at least two days to promote HSC survival and to facilitate their genetic modification by viral vectors. However, while growth factor signaling may support efficient gene correction of CD34 ⁺ cells, it may also contribute to partial differentiation, leading to loss of HSC properties. Adult HSCs are known to exist in a particular specific microenvironment that is in a hypoxic state (i.e., lack of oxygen) and that is involved in maintaining the functional properties of HSCs.

In addition, in vitro culture of human CD34 ⁺ cells under hypoxic conditions helps maintain HSC properties. Indeed, hypoxia can maintain Reactive Oxygen Species (ROS) of HSCs in an extremely low state. These highly reactive molecules induce oxidative stress and damage cells at too high levels, but at the same time can also act as signaling molecules driving HSC differentiation and proliferation.

A variety of cellular mechanisms are available for scavenging reactive oxygen species generated by physiological signals and various external pressures. In particular, several enzymes limit the accumulation of highly reactive substances by synergism, the superoxide dismutase (SOD) family converts O ₂ ^- to H ₂O₂, and then other different families of enzymes (e.g., thioredoxin, peroxiredoxin, catalase, or glutathione peroxidase) can metabolize H ₂O₂ to prevent its conversion to HO ^●. These systems are critical because elevated levels of reactive oxygen species in HSCs can lead to loss of differentiation and function, thereby reducing hematopoietic reconstitution capacity in vivo. When CD34 ⁺ cells (including HSCs) are used in the culture process of gene therapy, there is a potential for partial loss of function, mainly due to elevated levels of reactive oxygen species caused by non-physiological O ₂ levels and growth factor signaling. Under such culture conditions, the function of HSCs can be maintained by a variety of methods.

Recent studies have proposed a variety of HSC culture protocols, including culture under hypoxic conditions, culture in hydrophobic hydrogels, and culture in environments where small molecules such as UM171.25-27 are present. Although these methods of delivery are very attractive, hypoxic conditions and hydrogels are not easily regulated in clinical applications and the mechanism of action is not yet defined. In earlier studies, antioxidant pretreatment was found to protect HSCs from reactive oxygen species induced by low dose radiation. Based on this hypothesis, in vitro antioxidant treatment of HSCs prior to gene therapy may help maintain their function.

Bohemine is a purine analog, also a synthetic selective CDK inhibitor, with IC ₅₀ (Half maximal inhibitory concentration, semi-inhibitory concentration) for Cdk2/cyclin E, cdk2/cyclin A and Cdk9/cyclin T1 being 4.6. Mu.M, 83. Mu.M and 2.7. Mu.M, respectively. Bohemine can also inhibit ERK2, with corresponding IC ₅₀ at 52 μm, with less inhibition on CDK1, CDK4 and CDK 6. In order to achieve the goal of maintaining the self-renewal function of human HSC during in vitro culture, bohemine is used to protect HSC from oxidative stress damage generated during in vitro culture and to resist the influence of radiation.

Disclosure of Invention

To overcome the above-described deficiencies of the prior art, the present invention demonstrates Bohemine that is capable of protecting HSCs from oxidative stress during culture, maintaining their resting state in vitro culture, and maintaining the dryness of HSCs. It was also demonstrated that the addition of Bohemine to the culture medium in a cell/gene therapy regimen helped improve functional maintenance of HSCs in vitro culture.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the invention provides the use of Bohemine to maintain a resting state of functional characteristics of human Hematopoietic Stem Cells (HSCs) in vitro culture.

The second aspect of the invention provides a treatment method for maintaining the in-vitro culture functional characteristic resting state of human hematopoietic stem cells, which comprises the steps of inoculating human Hematopoietic Stem Cells (HSC) into a transduction medium before long-term culture, adding Bohemine and pre-culturing for 2 days, wherein the transduction medium is prepared by adding 80-150ng/mL Stem Cell Factor (SCF), 90-130ng/mL FMS-like tyrosine kinase 3 ligand (FLT 3L), 50-70ng/mL interleukin-3 (IL-3) and 7-15nM Thrombopoietin (TPO) into BIT medium.

Preferably, the Bohemine is used at a working concentration of 200-500 μm.

Preferably, in the long-term culture of human hematopoietic stem cells, STEMSPAN SFEM medium is substituted for BIT medium.

Preferably, the artificial blood stem cells are derived from umbilical cord blood.

Although the study of the present invention focused primarily on HSCs in early post-natal cord blood samples, the method may also be applicable to HSCs in different stages of development (more mature) in bone marrow or peripheral blood. Indeed, elevated levels of reactive oxygen species are generally associated with HSC aging, and it is well known that adult HSCs are less efficient in hematopoietic reconstitution than neonatal umbilical cord blood HSCs. Given the wide range of applications of bone marrow or mobilized peripheral blood cd34+ cells in autograft and gene therapy protocols, protecting these HSCs from further oxidative stress would help optimize the gene therapy protocol. Overall, these results demonstrate that Bohemine has an promoting effect on HSC maintenance and verify its effectiveness in protecting HSCs in vitro culture.

More preferably, mononuclear cells in umbilical cord blood are separated by Ficoll gradient centrifugation, and CD34 ⁺ cells are purified by immunomagnetic separation using a CD34 microbead kit, thereby obtaining the human hematopoietic stem cells.

In a third aspect, the invention provides the use of human hematopoietic stem cells pre-cultured by the treatment method of the second aspect in the manufacture of a medicament for the treatment of Metachromatic Leukodystrophy (MLD).

The ideal MLD treatment scheme must ensure that the therapeutic ARSA transgene achieves targeted high expression, and the expression mode needs to meet two conditions simultaneously, namely, maintaining the copy number of the wild type gene and maintaining the undifferentiated resting state of HSC in vitro culture, thereby avoiding the unexpected integration of the transgene and the low activity expression of ARSA enzyme. Based on Bohemine pretreatment HSC technology, a feasible scheme is provided for the high expression of targeted genes of specific cell types in the biomedical field. The potential of this technology has been explored for many clinical and preclinical studies of Hematopoietic Stem and Progenitor Cells (HSPCs) and T cells, and targeted transgene integration was successfully achieved at cellular endogenous gene sites through the Homology Directed Repair (HDR) pathway. It is known that over 200 different ARSA mutations can lead to good MLD, so that an ideal therapeutic regimen must employ a universal gene correction strategy to achieve a therapeutic effect that is independent of the type of mutation.

Therefore, the invention adopts a stepwise research method, firstly, hematopoietic Stem Cells (HSC) are pretreated by Bohemine to maintain the dryness of in vitro culture, then HSC are transfected, sgRNA with highest editing efficiency (87%) is screened out from the HSC, the transgenic integration efficiency (> 40%) of endogenous ARSA loci of the HSC under the screening-free condition is evaluated, the transgenic integration rate is improved, the expression of ARSA enzyme is optimized and improved, and a feasible treatment method is provided for MLD patients.

The difference in integration efficiency between Green Fluorescent Protein (GFP) and ARSA transgenes highlights the importance of HSC to maintain resting state in vitro culture. The study found that the frequency of ARSA integration observed from patient-derived HSCs was lower, which has an important correlation with long-term cryopreservation (> 10 years) of HSCs and reduced quality compared to freshly isolated donor cells. According to the invention, through careful design and adopting Bohemine pretreatment of HSC, the functional characteristics of in-vitro culture of the HSC are improved, the resting state is maintained, meanwhile, the ROS level and oxidative stress reaction are reduced, the cultured HSC treated by Bohemine shows better hematopoietic reconstitution capability in vivo, long-term in-vitro and in-vivo functions are reserved, the in-vitro functions and resting state of the HSC are maintained by inhibiting the differentiation of the HSC cells, the mitochondrial activity is delayed, the expression of ARSA enzyme is optimized and improved, and the expression level of the ARSA enzyme is equivalent to that of differentiated myeloid precursor cells of healthy adults.

In summary, the present study provides a viable treatment for MLD patients, whose efficacy has been demonstrated and has potential for laboratory to clinical conversion. This proof of concept study further provides a specific mutation independent treatment regimen for MLD patients, whose efficacy has also been demonstrated, with laboratory to clinical conversion potential.

Preferably, the medicament further comprises pharmaceutically acceptable excipients.

More preferably, the auxiliary materials comprise at least one of excipient, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrating agent, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, aromatic, anti-adhesive agent, integrating agent, permeation enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, inclusion agent, humectant, absorbent, diluent, flocculant and deflocculant, antioxidant, adsorbent, filter aid, and release retarder.

Preferably, the dosage form of the medicament comprises a tablet, capsule, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent or suppository.

Compared with the prior art, the invention has the beneficial effects that:

The invention provides a breakthrough treatment scheme for MLD, and by means of hematopoietic stem cell transplantation gene therapy optimized by small molecule inhibitor Bohemine, the ARSA enzyme activity of a patient can be obviously recovered, the level of healthy adults can be reached, and the abnormal metabolism of sulfate and neurodegenerative diseases can be effectively improved.

Bohemine can maintain its resting state during in vitro culture of HSC, avoiding elevated ROS levels and loss of self-renewal function due to culture stress. The HSC pretreated by the method has obviously enhanced clonogenic capacity and xenograft reconstitution capacity, still approaches to the characteristics of fresh HSC after short-term culture, and solves the problem of stem cell functional defects caused by in vitro culture.

The therapy optimizes the HSC in-vitro culture condition, improves the transgene integration rate and ARSA enzyme expression, enhances the treatment stability and success rate, and has strong clinical transformation potential. Meanwhile, scientific basis is provided for optimizing the HSC culture system, the application development of hematopoietic stem cells in the fields of cell therapy and gene therapy is promoted, and the method has great significance for MLD therapy and related research.

Drawings

FIG. 1 is a graph showing the relationship between Hematopoietic Stem Cell (HSC) in vitro culture function and molecular mechanism, wherein A is an experimental design flow chart, B is LTC-IC experimental function verification, C is principal component analysis, D is volcanic diagram, E-F (heat diagram+GSEA analysis), G is an epigenetic heat diagram, and H is a pathway GSEA analysis.

FIG. 2 is a graph of experimental results demonstrating Bohemine the antioxidant and antioxidant stress protective effects of Hematopoietic Stem Cells (HSC), A flow results (left) and scatter plots (right) of direct ROS levels, B antioxidant gene expression profiles, C flow histograms (left) and histograms (right).

FIG. 3 is a graph showing the results of experiments for verifying Bohemine functional protection of Hematopoietic Stem Cells (HSC), A primary CFU-C, B secondary CFU-C, C (5 weeks), D (10 weeks) by limiting dilution to count "Long term culture initiating cells (LTC-IC) frequency", E "human chimeric rate", F "myeloid/lymphoid differentiation ratio" was analyzed, and G "HSC phenotype cell ratio" was examined.

FIG. 4 is a graph showing experimental results of Bohemine in-vitro functional mechanisms of Hematopoietic Stem Cells (HSC), A. HSC growth curve was measured, B. Analysis of "immature surface marker (CD 34 ⁺CD90⁺)", C. Differentiation rate by CFSE staining, D-E. Cell cycle observation, F-G. Mitochondrial status was studied.

FIG. 5 is a graph of experimental results demonstrating the effect of gene editing combined with Bohemine pretreatment on Hematopoietic Stem Cell (HSC) gene correction efficiency and function, A: flow scatter plot, B: quantification of eGFP ⁺ ratio at different MOI, C: ddPCR detection of HDR (homology directed repair) signal plot, D-E: quantification of HDR ratio for different treatment groups, F: detection of "ARSA mRNA" by qPCR, G: ARSA enzyme activity, H: myeloid differentiation analysis.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

Metachromatic Leukodystrophy (MLD) is a rare genetic disease caused by mutations in the ARSA gene. This enzyme plays a key role in sulfate metabolism in brain cells, and its lack can lead to neurodegenerative disorders. The present invention provides an allogeneic hematopoietic stem cell transplantation (allogeneic-HSCT) gene therapy (autologous-HSCT) which maintains functional characteristics resting state of human hematopoietic stem cells in vitro culture with the aid of small molecule inhibitors, and which can be used as a potential therapeutic regimen for MLD, and which can significantly enhance recovery of ARSA enzyme activity (increase > 30-fold) in clinical treatment to levels comparable to healthy adults. In summary, the studies of the present invention provide a proven effective treatment for MLD patients, and the method has a strong clinical transformation potential.

In the resting state, the level of Reactive Oxygen Species (ROS) in human Hematopoietic Stem Cells (HSCs) is extremely low. Under stress conditions, HSCs are activated and initiate proliferation and differentiation procedures to ensure regeneration of blood cells. Once activated, HSCs raise ROS levels and act as signaling molecules regulating their proliferation and differentiation processes. However, after stress ends, the ROS levels of HSCs need to be restored to normal, otherwise HSC depletion may result. Studies have shown that small molecule inhibitors can prevent loss of HSC self-renewal function and maintain their resting state in vitro in a variety of situations. For example, fresh cord blood HSCs (D0 CB HSCs) are known to be mostly in a quiescent state, characterized by a slow cell division differentiation process, delayed mitochondrial activation, while maintaining HSC self-renewal potential and without the occurrence of HSC depletion. This phenomenon suggests that small molecule inhibitors can be used to maintain the self-renewing functional properties of HSCs under in vitro culture induced stress conditions, keeping them in a resting state.

HSCs are increasingly used in cell and gene therapies, which typically require in vitro culture of HSCs for days. It was found that even a short in vitro culture time of HSCs resulted in severe defects in their self-renewing function properties-their transcription program was shifted from stem cell properties to differentiation direction, failing to maintain resting state, consistent with the expected effect. In addition, an interesting phenomenon was observed in HSC in vitro culture experiments, in which after pretreatment of in vitro cultured HSC with small molecule inhibitor Bohemine, its clonogenic capacity was significantly improved in the secondary clonogenic unit cell (CFU-C) assay, and its reconstitution capacity was also significantly enhanced in xenograft models, and both of these performances were superior to untreated in vitro cultured HSC. In summary, the study of the present invention demonstrates that the addition Bohemine can protect the self-renewing functional properties of HSCs during in vitro culture, maintaining their resting state.

The results of the present study demonstrate that Bohemine is capable of protecting HSC function in short term (2 day) in vitro cultures, which induce a shift in HSC status to progenitor cell characterization, which activates HSCs, with a gradual loss of self-renewal capacity and stem cell characteristics. Bohemine can enhance the generation of CFU-C, increase the occurrence frequency of long-term culture initiating cells (LTC-IC), and save the in vivo hematopoietic reconstitution capacity loss caused by culture, so that the characteristics of the cultured HSC are closer to fresh 0-day HSC (D0-HSC).

The present invention has found that even a short incubation time results in a severe impairment of the self-renewal function properties of HSCs, manifested by increased ROS levels. In addition, studies have demonstrated that small molecule inhibitors Bohemine are able to protect HSCs from culture-induced stress, maintain their self-renewing functional properties, and maintain in vitro culture resting states. These results lay the foundation for optimizing the culture conditions of HSCs. By optimizing the culture conditions, the integration rate of the transgenes can be improved, the expression of ARSA enzyme is further enhanced, and an effective treatment method is finally provided for MLD patients.

The present invention will be described in detail below with reference to specific embodiments thereof in order to fully and clearly demonstrate the technical aspects of the present invention and the significant advantages thereof.

1. Experimental method

1.1 Umbilical cord blood sample and in vivo experiments

Umbilical Cord Blood (CB) samples were collected from healthy infants and cooperatively collected from the Guangdong cord blood hematopoietic stem cell bank, and written informed consent of the mother had been obtained prior to collection.

Purification of hematopoietic Stem cells (CD 34 ⁺ cells) the standard procedure was followed by first separating monocytes in blood using Ficoll gradient centrifugation, and then purifying CD34 ⁺ cells by immunomagnetic bead sorting using CD34 microbead kit (Miltenyi Biotech, paris, france), i.e., hematopoietic Stem cells. The treated hematopoietic stem cells can be used directly for experiments or frozen in serum containing 10% DMSO (placed in liquid nitrogen) and thawed for subsequent experiments.

1.2 Cell culture

Bohemine A complete medium for cell transduction was formulated from BIT medium (STEMCELL Technologies) supplemented with 100ng/mL Stem Cell Factor (SCF), 100ng/mL FMS-like tyrosine kinase 3 ligand (FLT 3L), 60ng/mL interleukin-3 (IL-3) and 10nM Thrombopoietin (TPO) (all available from PeproTech, USA). In subsequent long term Hematopoietic Stem Cell (HSC) culture experiments, the BIT medium was replaced with STEMSPAN SFEM medium (STEMCELL Technologies).

Drug and treatment:

The drugs used in the experiments were Bohemine (300. Mu.M), hydrogen peroxide (H ₂O₂, 100. Mu.M), vascular endothelial growth factor A (VEGFA, 50 ng/mL), ZM32388 (10 nM), brivanib (Brivanib, 50 nM). Before the initiation of the long-term culture, hematopoietic stem cells were pretreated with 300 μ M Bohemine, i.e., bohemine was incubated with hematopoietic stem cells for 2 days.

1MM Bohemine A mother liquor was prepared by dissolving 1mg Bohemine in 2.9375mL of dimethyl sulfoxide (DMSO). The mother solution is stored in a refrigerator at-80 ℃ after being packaged, so as to avoid repeated freezing and thawing, and is diluted to the concentration of the working solution when in use.

1.3 Flow cytometry

Cell surface staining procedures were performed at room temperature in the dark, after staining, the cells were washed with PBS and resuspended, and the staining used Hoescht and ZombiAqua (Biolegend) as viability markers.

Intracellular p38MAPK staining was performed using the phosphoflow staining method (BD Biosciences), following the manufacturer's instructions, by first fixing the cells with Fix Buffer I (BD Biosciences) at 37℃for 10 minutes, followed by permeabilizing the cells with Perm Buffer II (BD Biosciences) on ice for 1 hour, followed by intracellular staining in PBS containing BSA and EDTA.

Cell analysis is typically performed on a BD Canto II or BD LSRII SORP flow cytometer, and cell sorting is performed by BD Influx or BD Aria III SORP flow cytometer. All experimental results were analyzed using FlowJo software.

1.4 Colony forming unit cell (CFU-C) assay

CFU-C cultures were initiated with 500 bead-sorted Hematopoietic Stem Cells (HSCs) that had been pre-cultured with Bohemine for 2 days (no pre-treatment as control) at 37 ℃ in a CO ₂ incubator, with 3 replicates per set of experiments. The specific procedure is as follows, the HSC described above is inoculated into methylcellulose medium H4435 (H4435, STEMCELL Technologies), cultured for 12-14 days, and the colonies formed are identified and counted. Plates of similar colony numbers and types were then resuspended in 25℃pre-warmed PBS buffer and 1% of the total recovered cells were inoculated into the secondary culture system.

1.5 Long-term culture initiating cell (LTC-IC) assay

After 2 days of pre-incubation with Bohemine (no pre-treatment as control), hematopoietic Stem Cells (HSCs) were seeded in a limiting dilution method in 96-well plates (using Myelocult medium, H5100, STEMCELL Technologies) pre-plated with MS5 stromal cells, and long-term colony counting-co-immune culture experiments (LTC-IC experiments) were performed using Myelocult medium, with the specific steps of cells continued to be incubated for 5 weeks with half volume of medium changed weekly, cells were recovered after the end of incubation, inoculated in 500 μl of methylcellulose medium (H4435), and colony growth was observed and assessed after 10-12 days.

For prolonged Long Term Culture (LTC) experiments, hematopoietic stem/progenitor cells (HSPC) were cultured in 6-well plates for 5 weeks, again with half the volume of medium replaced weekly, CD34 ⁺ cells (HSC cells) were sorted after the end of the first 5 weeks culture, and plated in 96-well plates with different concentrations of limiting dilution.

1.6 Transplantation experiments

NOD. Cg-Prkdc (scid) Il2rg (tm 1 Wjll)/SzJ (NSG) mice were housed in pathogen free animal facilities. Adult NSG mice of 8-12 weeks of age were selected and irradiated with a sublethal dose of 2.5 Gy using a GSRD-irradiator followed by anesthesia with isoflurane prior to intravenous injection of human cells. All experimental procedures strictly follow the ethical specifications of animals and meet the requirements of the local ethical committee.

Hematopoietic stem cells (CD 34 ⁺ cells) were extensively cultured in transduction medium for 2 days after pretreatment (or no pretreatment) (treatment group was treated with Bohemine at a final concentration of 300 μm, control group was not treated). After counting the cells, hematopoietic stem cells were intravenously injected into the irradiated mice, each of which was injected at an amount of 1-2.5X10 ⁴ cells.

Mice were sacrificed 16 weeks after transplantation, then 4 long bones were removed for bone marrow analysis, and blood samples were collected for related detection.

1.7 Microarray transcriptional analysis

Transcriptome analysis uses Affymetrix human Clariom D chips, using 3 sorted Hematopoietic Stem Cell (HSC) samples from different cord blood under each experimental condition.

Data analysis was done by TAC software, GSEA (gene set enrichment analysis) and molecular characterization analysis software. The judgment standard of the differential expression gene is that the gene expression change multiple is more than or equal to 2 and the P value is less than or equal to 0.05. Thermal mapping was accomplished by means of http:// shinyheatm ap. The relevant experimental data has been uploaded to the Array Express database (number: E-MTAB-12121).

1.8 Measurement of ROS level and mitochondrial activation

ROS levels were measured on day 0 (D0), day 1 (D1) and day 2 (D2) following Bohemine treatment (i.e. Bohemine co-incubation with hematopoietic stem cells for 2 days), as follows:

cells were stained with surface antibody, stained with CellRox DeepRED dye, incubated at 37 ℃ for 30min, washed, fixed with BD cell fixation buffer, and then analyzed on BD Canto II flow cytometer. To verify a positive control for high ROS levels, a portion of the cells were incubated with TBHP for 30 minutes, followed by the addition of CellRox DeepRED probes.

Cells were stained with surface antibody, incubated with CellRox Orange dye at 37 ℃ for 30min, washed and incubated on ice, immediately analyzed by BD Canto II flow cytometer. If ROS are to be induced, cells are pre-treated with Bohemine hours, ROS can be prepared by incubation with TBHP for 30 minutes prior to staining CellRox Orange, or by addition of 100. Mu. M H ₂O₂ for the last 15 minutes of staining CellRox Orange.

Mitochondrial activation was measured by staining cells with surface antibodies, mixing with 50. Mu.M TMRE (tetramethylrhodamine ethyl ester) and 50nM MTG (mitochondrial Green fluorescent Probe) in PBS, incubating at 37℃for 30min, and immediately analyzing on BD Canto II or LSR-SORP flow cytometry.

1.9 Expression of antioxidant Gene or antioxidant Pattern

The antioxidant spectrum is defined by detecting the relative expression quantity of transcripts of 21 key antioxidant genes through a real-time quantitative PCR technology (the detection method is defined by adopting a Roche application science company LightCycler 480 microplate cycler platform in reference to a ：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.）. experiment, and carrying out real-time fluorescent quantitative PCR analysis by combining a universal probe library designed by probe discovery software, wherein the relative expression quantity of the antioxidant genes is calculated and analyzed through a2 ^-ΔΔCt method, and finally the overall outline of the antioxidant spectrum is presented.

1.10 Preparation of lentiviral vector ARSA LV

The ARSA LV used in this study was prepared by transient transfection of 293T cells with four plasmids prepared by GeneArt company (ThermoFisher Scientific) in vitro synthesis. First, 293T cells were inoculated into T162 flasks and expanded in Dulbecco's modified Eagle's medium (DMEM; biochrom)) and subsequently transferred to a 10-well plate Opti-MEM medium (Thermo FISHER SCIENTIFIC) for culturing in a cell factory. Four plasmids encoding the two core packaging constructs (pKLGag/pol and pKRev), the envelope construct (pK.G) and the transfer vector construct (pARSA), respectively, were then transfected with JetPEI transfection reagents (Polyplus transfection, thermo FISHER SCIENTIFIC). After 24 hours of co-incubation of 293T cells transfected in Opti-MEM medium, 293T cell supernatants were collected and stored at 4 ℃. The 293T cells were then replaced (the adherent 293T cells were rinsed with DMEM medium followed by addition of 10% fetal bovine serum (Gibco) and 1% L-glutamic acid (Biochrom) in DMEM medium) and the 293T cells were further cultured for 24 hours, and a second 293T cell supernatant was collected. After mixing the two collected supernatants, virus concentration was performed by centrifugation at 280,000Xg for 4 hours in a 4℃ultracentrifuge. The concentrated viral pellet is resuspended in Opti-MEM medium, and after mixing, 0.2 μm filter sterilization and aseptic filling are performed, and finally the purified lentiviral vector is stored in an ultra-low temperature environment at-80 ℃.

1.11, Flow analysis

CD34 ⁺ HSC were enriched using a magnetically activated cell sorting system (CLINIMACS SYSTEM; miltenyi Biotec). Enriched HSC purity from healthy donors and post-bone marrow transplant lymphocytic disease (MLD) patients was >90% in all isolated samples. 28 CD34 ⁺ HSCs were cultured in StemMACS hematopoietic stem cell expansion medium (Miltenyi Biotec) supplemented with human stem cell factor (SCF; 100 ng/mL) and interleukin-3 (IL-3, 100 ng/mL; miltenyi Biotec) at 37℃for 3 days before culture in 5% CO ₂.

Subsequently, a two-stage myeloid differentiation protocol was used. 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 medium. From day 6 to 9 (second stage), M-CSF alone (50 ng/mL) was added, and the maturation of myeloid cells was finally monitored by flow cytometry. Cell analysis (BD FACSCalibur) was performed on day 10 using FITC-conjugated anti-CD 33 (Miltenyi Biotec), PE-conjugated anti-CD 14 (Miltenyi Biotec), perCP-conjugated anti-CD 45 (Miltenyi Biotec), APC-conjugated anti-CD 11b (Miltenyi Biotec) and APC-conjugated anti-CD 66b (Miltenyi Biotec).

1.12, QPCR and ddPCR

Viral titers were determined by quantitative polymerase chain reaction (qPCR) and drop digital PCR (ddPCR). To obtain viral DNA after LV capsid disruption, 2 mL concentrated viral supernatant was taken and digested with 2IU DNase I (NEB, thermo FISHER SCIENTIFIC) to a final reaction volume of 40. Mu.L. Samples were incubated at 37℃for 30 min, then treated at 75℃for 15min, and tested using proteinase K (1. Mu.L; qiagen) added to 5. Mu.L of DNase I digest to a final volume of 20. Mu.L, and incubated at 50℃and 98℃for 30 min and 10 min, respectively.

QPCR was performed using a CFX real-time PCR instrument (Bio-Rad), the reaction system containing 2. Mu.L of proteinase K digestion product, 12.5. Mu.L of KAPA Probe FAST (Bio-Rad), 1. Mu.L of ITR primers (5. Mu.M) and ITR Probe (5. Mu.M) each, and 2.5. Mu.L of water, with the procedure set to 95℃for 3 minutes followed by 40 cycles of 95℃for 3 seconds and 60℃for 20 seconds.

The ddPCR reaction system (Bio-Rad) was prepared in a total volume of 20. Mu.L, and the specific components were ddPCR multiplex premix (13. Mu.L), 950 nM primer (1. Mu.L), 250 nM Probe (1. Mu.L) and 350 ng DNA template (5. Mu.L). Simultaneously, ddPCR was performed on a C1000 Touch thermocycler (Bio-Rad) for 10 minutes at 95℃for 30 seconds at 95℃for 40 cycles, 57℃for 1 minute and 72℃for 2 minutes, and finally enzyme inactivation was performed at 98℃for 10 minutes. The primer and probe information is detailed in Table 1.

TABLE 1 oligonucleotide sequences for qPCR and ddPCR

Note that ：[1].Mern DS, Thome´ C. Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders. BMC Musculoskelet 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 gene correction 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.

The specific experimental procedure is as follows:

(1) The DNA samples were split into about 20,000 droplets using a QX200 droplet digital PCR (ddPCR) generator (Bio-Rad) and subsequently transferred to a 96-well plate;

(2) Sealing the 96-well plate using a PX1 PCR plate heat sealer (Bio-Rad) to prevent evaporation of the liquid during the reaction;

(3) The sealed 96-well plate is placed on a C1000 Touch thermal cycler (Bio-Rad) for PCR reaction, the reaction procedure is that the initial denaturation is carried out for 10 minutes at 95 ℃, 40 cycles are carried out (30 seconds at 95 ℃,1 minute at 61 ℃ and 2 minutes at 72 ℃) and finally the enzyme inactivation is carried out for 10 minutes at 98 ℃;

(4) The reaction products were detected by a QX200 droplet analyzer and the data were processed using QuantaSoft v1.6.6 analysis software (Bio-Rad).

1.13、qRT-PCR

On day 10 post-HSC transfection, total RNA was extracted using RNEASY MINI KIT (Qiagen) followed by cDNA synthesis using QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR detection is carried out by CFX96TM real-time fluorescent quantitative PCR system (Bio-Rad) and is matched with KAPA SYBR FAST 2X MasterMix (KAPA Biosystems) to finish cDNA amplification and quantification.

The experimental results were normalized with the expression level of β2 microglobulin (β2m) as an internal reference, and the Crossover Point (CP) value of the unknown sample was calculated by equation 2 (CP _β2M-CP_{target gene}）. QRT-PCR primers used in this experiment were specific (primer source reference ：Johnson RL, Milenkovic L, Scott MP. In vivo functions of the patched 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.）, only detected ARSA transcripts (transgene specificity), and did not cross-react with the expression of endogenous ARSA).

For samples not transduced (i.e., control), no signal was detected in the qRT-PCR analysis, so the Ct value was set to the maximum number of cycles of the experiment (40 in this experiment) to calculate fold change. This process is consistent with the method of multiple analysis software (e.g., applied Biosystems DATAASSIST V3.0.0 and Integromics REALTIME STATMINER), i.e., the maximum Ct value assignment is used for undetected values. It should be noted that this method may result in a bias in the actual fold change (i.e., overestimated), but can be used to map the relative amount of mRNA expressed in Bohemine + Lentivirus (LV) treated samples relative to control.

1.14, ARSA enzyme Activity assay

The quantitative detection of the ARSA (arylsulfatase A) enzyme activity adopts an artificial substrate to carry out a functional experiment on nitrocatechol sulfate (pNCS; merck), and the specific method refers to the specification of a manufacturer and comprises the following steps:

(1) Sample preparation cell lysates were prepared at a concentration of 2.5X10 ⁶ cells/ml.

(2) The reaction system comprises mixing cell lysate with substrate solution, wherein the substrate solution comprises 10mM pNCS, 0.5mM sodium pyrophosphate (sodium pyrophosphate) and 10% NaCl, and dissolving in 0.5M sodium acetate buffer (pH 5.0).

(3) Incubation reaction the mixed reaction system was incubated at 8 ℃ for 48 hours.

(4) After the reaction is finished, 0.5M NaOH is added to stop the reaction, and the absorbance value is measured at the wavelength of 514nm by a spectrophotometry method, so that the conversion rate of a substrate is reflected, and the ARSA enzyme activity is quantified.

The method directly reflects the functional activity of the ARSA enzyme by detecting the conversion efficiency of the artificial substrate, and is a key experimental means for evaluating the biological function of the enzyme.

1.15 Statistical analysis

Data are expressed as mean ± standard deviation. Statistical significance was determined by t-test or Fisher exact test between the two groups and one-way anova between the three groups.,,A kind of electronic deviceIs considered to be statistically significant. Statistical analysis was performed using version GRAPHPAD PRISM 6.01.01. Limiting dilution assays in vivo and in vitro were performed using L-Calc software.

2. Experimental results

2.1 In vitro culture causes changes in HSC characteristics and HSC Gene expression function

To understand the effect of in vitro culture on functional properties of HSCs, limited dilution LTC-IC (an experimental model to assess HSC immature function) experiments were performed. The experimental results are shown in figure 1, wherein A is an experimental design flow chart showing research frameworks from HSC sorting to grouping from 'non-cultured (D0-HSC)' to 'cultured for 2 days (D2-HSC)' and then to functional experiments (LTC-IC) and molecular experiments (microarray), B is the verification of the functions of the LTC-IC experiments, the stem cell activity difference (D2 activity is lower) of the generated colony is quantified by limiting dilution method statistics, 'C is the principal component analysis, the cluster separation of the D0/D2-HSC is shown, the integral difference of the two groups of gene expression profiles is totally different', D is a volcanic map, screening differential genes, red blue spots represent significant up-down-regulated genes, the gene expression change scale (5476 differential genes) caused by 'cultured for 2 days', E-F (heat map+GSEA analysis) focuses on the expression difference of the characteristic genes of the generated colony, GSEA is further enriched in D0, the gene is enriched in D2, the differential gene is enriched in the G is enriched, and the differential pathway is enriched in the differential pathway is shown by the differential map, and the differential pathway is controlled by the differential map.

First, human HSCs were purified by cell sorting according to the CD34 ⁺CD38^-CD45RA^-CD90⁺ phenotype, either without seeding directly under LTC-IC conditions (D0-HSCs (non-cultured HSCs)) or with seeding of D2-HSCs (HSCs on day 2 of culture, A in FIG. 1) to LTC-IC conditions 2 days after culture. Subsequently, the cell frequency of colonies generated under LTC-IC conditions was compared for D2-HSC with non-cultured HSC (D0-HSC). Under LTC-IC conditions, D2-HSC were at a frequency of (1/9,95% CI [1/11-1/8 ]) under LTC-IC conditions, while D0-HSC were at a frequency of (1/4,95% CI [1/5-1/4 ]), D2-HSC were able to colonise cells at a frequency 2.25 times lower than D0-HSC after 5 weeks, indicating that D2-HSC lost the HSC stem cell characteristics during culture (B in FIG. 1). To further understand the molecular mechanisms of these functional changes, transcriptome analysis was performed using CLARIOM D microarray technology and the transcriptional profiles between D2-HSC and D0-HSC were compared. Principal component analysis showed that D2-HSC and D0-HSC clustered separately (C in FIG. 1), revealing different transcriptome maps. In total, 5476 genes were differentially regulated under both conditions, with 1868 genes down-regulated in D2-HSC and 3608 genes up-regulated in D2-HSC (D in FIG. 1). First, a Gene Set Enrichment Analysis (GSEA) (E-F in FIG. 1) was performed using some C2-CGP HSC and progenitor cell specific gene sets in https:// www.gsea-msigdb: org/GSEA/msigdb/index. HSC and progenitor gene signatures in D2-HSC were found to be down-regulated and up-regulated, respectively, compared to D0-HSC, confirming induction of HSC differentiation and resulting loss of HSC function during 2 days of culture in vitro (E-F in FIG. 1). In addition, genes involved in epigenetic modification, such as TET2, DNMT3B, EZH1 and EZH2, were found to be differentially expressed when comparing D2-HSC and D0-HSC conditions, and were specifically expressed as increased expression of EZH2 in D2-HSC, which is associated with activation and differentiation of HSC, while decreased expression of EZH1, which is associated with the dry character of HSC. Similarly, TET2 expression is also lower in D2-HSC than in D0-HSC (G in FIG. 1). Finally, this analysis shows that in HSCs, culture induced cell cycle/differentiation procedures and oxidative stress persist for 2 days. When using GSEA software for gene ontology analysis, the marker pathways enriched after 2 days of culture are related to cell cycle and oxidative phosphorylation. In contrast, the features of enrichment in D0-HSCs are associated with hypoxia (H in FIG. 1). Indeed, HSCs have been reported to exhibit molecular characteristics related to hypoxia, regardless of oxygen concentration. Thus, in vitro culture not only drives changes in HSC functionality, but also initiates metabolic switching.

2.2, Bohemine reduction of ROS and oxidative stress

To verify whether Bohemine can retain the potential of HSCs during the two-day stress culture. First, the antioxidant effect of Bohemine was examined in Hematopoietic Stem Cells (HSCs). The experimental procedure is to pretreat HSCs with Bohemine prior to inducing oxidative stress, then induce oxidative stress with t-butyl hydroperoxide (TBHP), and then detect Reactive Oxygen Species (ROS) levels with CellRox orange probe. The results show Bohemine can inhibit TBHP-mediated elevation of ROS levels in human HSCs, indicating that they have antioxidant effects. Thus, ROS levels in Bohemine-treated or untreated HSCs were further measured at D0 (not cultured) and day 2 of culture (D2). The experimental results are shown in fig. 2, wherein, A is a flow result of directly measuring ROS level, the left side is provided with a CellROX dark red probe, the fluorescence intensity (MFI) of a 'Bohemine treated group (light purple peak)' and an 'untreated D2-HSC group (gray peak)' are compared, the effect of reducing ROS is visually reflected Bohemine, the right side scatter diagram quantifies 'the increase of the multiple of ROS level', the inhibition effect of Bohemine on ROS is further verified, B is indirectly reflected by utilizing an 'antioxidation gene expression profile', the comparison of D0-HSC (dotted line and represents the standard of non-oxidative stress), the antioxidation gene expression of D2-HSC (black bold line and represents the state under oxidative stress), the graph of Bohemine treated D2-HSC (blue line) is close to D0 after Bohemine treatment, the antioxidation gene is regulated and released, the difference of the phosphorylation p38 level of the left side flow type comparison of the C and the quantification of untreated D2-HSC group (gray peak) is proved, and the reduction of MAPK is demonstrated by a right side columnar diagram, and the reduction of MAPK is indirectly prevented from oxidative stress.

The results show that under untreated conditions, ROS levels for HSCs are on an upward trend from D0 to D2. In addition, ROS levels tend to decrease when cells are treated with Bohemine (a in fig. 2). To enhance these results, the antioxidant effect of Bohemine on HSCs was assessed after 2 days of culture using an "antioxidant map" test. The "antioxidant map" test provides a comprehensive view of the activation of multiple antioxidant pathways with increased intracellular ROS levels by tracking the expression levels of key antioxidant gene transcripts. Many antioxidant genes were significantly over-expressed in D2-HSC compared to non-cultured cells (D0-HSC), confirming that D2-HSC was indeed affected by oxidative stress compared to D0-HSC (B in FIG. 2, D2-HSC, D2-HSC shown in bold black lines, D0-HSC shown in dashed lines, contrast to see this difference). In contrast, the antioxidant gene expression profile in Bohemine-treated D2-HSC (B, bohemine D2-HSC, blue line in FIG. 2) shifted toward D0-HSC, indicating that many of the analyzed genes were down-regulated in the presence of Bohemine. To further demonstrate the role of Bohemine in inhibiting ROS production in culture, the phosphorylation status of the oxidative stress secondary messenger p38MAPK (p 38 mitogen-ACTIVATED PROTEIN KINASE, mitogen-activated protein kinase) was studied. After two days of culture, p38MAPK phosphorylation was significantly reduced in D2-HSC pre-treated Bohemine compared to untreated D2-HSC, but still present (C in FIG. 2), indicating a decrease in ROS levels following Bohemine treatment. These results above demonstrate that Bohemine can reduce ROS levels in D2-HSCs, bohemine pretreatment can protect HSCs from oxidative stress over a 2 day incubation period.

2.3, Bohemine treatment of cultured HSC with better hematopoietic reconstitution in vivo, preserving long-term in vitro and in vivo HSC function

The effect of Bohemine treatment on HSC function was studied, and CFU-C experiments were performed using D2-HSC and their immature functions were further explored by their re-culture capacity (continuous CFU-C assay). Briefly, HSCs, with or without Bohemine treatment in advance, were sorted, cultured in complete medium for 2 days, and then inoculated into CFU-C medium. The results of the experiment are shown in FIG. 3, wherein A is the "primary CFU-C experiment" which proves that Bohemine does not affect the initial colony formation of HSC, and B is the "secondary CFU-C experiment" which shows that Bohemine treated group colonies are more, which shows that the potential of colony formation of HSC can be maintained, and the function loss caused by culture can be prevented. C (5 weeks) and D (10 weeks) are subjected to limiting dilution method, statistics of 'long-term culture initiation cell (LTC-IC) frequency', bohemine treatment groups have higher LTC-IC frequency (close to D0-HSC), and prove that the treatment groups can protect HSC immature functions and maintain effects after prolonged culture, E statistics of 'human cell chimeric rate', bohemine treatment groups have higher chimeric rate and lower heterogeneity, which shows that the treatment groups promote the in vivo reconstruction efficiency of D2-HSC, F analysis of 'myeloid/lymphoid differentiation ratio', no obvious deviation, proof that Bohemine does not influence the differentiation direction of HSC, G detection of 'HSC phenotype cell ratio', and treatment groups have rising trend, and indirectly support the HSC protection function.

The results show that in primary culture, the number of colonies formed under both conditions was comparable (A in FIG. 3), while in secondary CFU-C culture, bohemine pre-treated D2-HSCs showed a significantly increased colony count compared to untreated D2-HSCs (B in FIG. 3). For two independent cord blood samples, the clonogenic capacity of HSC was measured and compared using D2-HSC (untreated D2-HSC and Bohemine-treated D2-HSC) and untreated D0-HSC, respectively. The untreated D2-HSC had a reduced secondary clonogenic capacity compared to the untreated D0-HSC, whereas Bohemine pretreated D2-HSC prevented this loss of function. In one experiment, three-stage CFU-C assays were performed and Bohemine-pretreated D2-HSC were found to generate more clones. This indicates that either the immature progenitor cells are better preserved or the self-renewal capacity of the HSCs is enhanced, with Bohemine pretreatment, with a greater number of CFU clones formed upon secondary (and tertiary) resurfacing. Furthermore, no deviation in CFU-C type was observed, demonstrating Bohemine did not affect HSC differentiation. To further confirm the effect of Bohemine on HSCs, the potential effect of Bohemine on the self-renewal potential of D2-HSCs was evaluated, and the frequency of LTC-ICs (long-term culture initiating cells, i.e., the least mature cells) was quantified by limiting dilution experiments. The results show that Bohemine pretreatment promotes maintenance of LTC-IC in D2-HSC, with a frequency close to that of D0-HSC (1/5,95% CI [1/6-1/5], C in FIG. 3) compared to untreated D2-HSC. To assess Bohemine maintaining the function of more immature HSC cells, a similar experiment was performed, including an extended (10 week) incubation period. After the end of the first 5 weeks of batch culture, CD34 ^⁺ cells that persisted in LTCs were sorted and inoculated with limiting dilutions for 5 weeks before their clonogenic capacity was assessed. After 10 weeks of incubation, untreated D2-HSC (1/98,95% CI [1/81-1/118]; D in FIG. 3) showed 50% higher LTC-IC frequency in Bohemine-treated D2-HSC (1/66,95% CI [1/55-1/79 ]), compared to Bohemine, which demonstrated the potential to protect HSC immature function.

To verify these results, CD34 ⁺ cells were pre-treated with Bohemine or not, cultured in complete medium for 2 days (D2-CD 34 ⁺ cells), and then injected in vivo in immunodeficient NSG mice (NOD-PRKDCSCIDIL 2rgem 1/Smoc). 16 weeks after transplantation, mice were sacrificed and human chimeras were detected using specific anti-human CD45 antibodies, and their long-term reconstitution potential was assessed. The results showed that D2-CD34 ⁺ cells produced human hematopoietic progenitors in vivo, but were less efficient than fresh CD34 ⁺ cells (D0-CD 34 ⁺) because of the high heterogeneity of hCD45 ⁺ cell levels detected upon transplantation of D2-CD34 ⁺ cells in NSG mouse bone marrow (E in fig. 3), which was reduced under the conditions of D2-CD34 ⁺ pre-treated with Bohemine. In fact, 86% of mice injected with Bohemine pre-treated D2-CD34 ⁺ cells showed human chimerism in a proportion >10%, while in mice injected with untreated D2-CD34 ⁺ cells this proportion was 57% (E in fig. 3). Neither myeloid nor lymphoid cell bias was detected (F in fig. 3). However, using classical HSC markers, a trend of higher HSC phenotype levels (although not significant; G in FIG. 3) was observed in the mouse bone marrow of D2-CD34 ⁺ cells receiving Bohemine pretreatment. In addition, one continuous implantation experiment was performed. There was no difference in the percentage of chimeras in the primary recipient mice, but Bohemine-pretreated D2-CD34 ⁺ cells were more potent in the secondary recipient, indicating that the function was better maintained when HSCs were subjected to Bohemine treatment during culture. taken together, these data indicate Bohemine that it is possible to preserve hematopoietic reconstitution function of HSCs during 2 days of culture (G in fig. 3).

2.4, Bohemine maintenance of HSC in vitro function by limiting HSC cell differentiation, ensuring HSC in vitro resting state and delaying mitochondrial Activity

To understand how Bohemine maintains HSC function in vitro, a number of parameters were monitored, such as proliferation, expression of immature cell surface markers, and cell division rate at different culture time points. The experimental results are shown in FIG. 4, wherein A is a curve for measuring HSC growth, which proves that Bohemine has no significant effect on "cell number increase", B is analyzed for "immature surface marker (CD 34 ⁺CD90⁺)", the marker runs off with prolonged culture time, but Bohemine treated group is not different from untreated group, C is lower in differentiation rate as seen by CFSE staining, bohemine treated group is lower in differentiation rate, which proves that it can "delay HSC differentiation", and D-E is in resting phase (G ₀ phase) for more HSC in Bohemine treated group by observing cell cycle. F-G study on mitochondrial status, F showed no obvious difference in mitochondrial quality, G found that Bohemine treated groups had lower mitochondrial membrane potential (activation delay) through TMRE probe, indicating that it had protective HSC function.

The results show that Bohemine was not found to have a significant effect on HSC cell growth (a in fig. 4). As expected, the HSC phenotype (CD 34 ⁺CD90⁺) gradually lost with prolonged incubation time, and no difference was observed between untreated and Bohemine-pretreated D2-CD34 ⁺ cells (B in fig. 4). However, differentiation rate (CFSE (carboxyfluorescein diacetate succinimidyl ester) staining) of Bohemine-pretreated HSC cells was reduced compared to untreated D2-HSC (C in fig. 4). Thus, the cycle status of HSC cells was examined after two days of culture. The Bohemine-pretreated D2-HSC were clearly more in a resting state, which accounts for the delay observed in CFSE cell differentiation experiments (D-4E in FIG. 4). Finally, mitochondrial membrane potential was measured by TMRE probe, mitochondrial mass was detected by MTG probe, and mitochondrial activation in Bohemine pretreated and untreated D2-HSC was analyzed. It can be observed that, despite the very limited effect of Bohemine on mitochondrial quality (F in fig. 4), mitochondrial activation was delayed (G in fig. 4) after 2, 4 and 7 days of culture in HSCs pretreated with Bohemine. In summary, the present invention demonstrates Bohemine that protects maintenance of HSC function in vitro by limiting cell differentiation rate and metabolic activation.

2.5, Bohemine pretreatment of HSC for the treatment of Metachromatic Leukodystrophy (MLD)

Lentiviral vector ARSA LV was transfected with healthy donor-derived Hematopoietic Stem Cells (HSCs) in DMEM medium of varying multiplicity of infection (MOI 2000, 1000 and 500, respectively) for 14 hours followed by culture in myeloid differentiation medium (StemMACS) immediately after gene editing for 10 hours. After 10 days of gene editing and lentivirus transduction, HSC gene correction efficiency was evaluated by various molecular analysis methods such as ddPCR, qPCR and ARSA enzyme activity detection, and flow cytometry analysis was performed on cells transduced with eGFP (GenBank: NG_ 009260.2) and ARSA (GenBank: U55762.1) donor templates. It is particularly noted that eGFP expression is only induced upon successful integration into the endogenous ARSA locus, a process that is regulated by the endogenous ARSA promoter. The experimental results are shown in FIG. 5, wherein A is a signal diagram for quantifying the HDR (homologous directional repair) in different treatment groups through a flow scatter diagram, the ratio of eGFP ⁺ cells in the 'Bohemine +LV group' is visually displayed to be far higher than that in a control group, B is a ratio of eGFP ⁺ in different MOI, the integration rate is proved to be more than 47% when MOI=1000, the effectiveness of a strategy is verified, C is a signal diagram for detecting HDR (homologous directional repair) through ddPCR, D-E is a ratio of HDR in different treatment groups, and corresponds to the flow result, the integration rate of the Bohemine pretreatment group is proved to be higher, F is detected through qPCR, the expression quantity of the Bohemine treatment group is 220 times higher than that of the control group, the effective transcription of an integrated gene is proved, G is ARSA enzyme activity, the gene editing/Bohemine treatment does not destroy the original functions of HSC, H is marrow differentiation analysis, CD33/CD11B/CD66B and other marrow markers are analyzed, and the ratio of each group is similar, and the Bohemine +LV treatment does not affect the differentiation direction of the HSC.

Remarkably, the results show that the strategy of the invention is capable of integrating eGFP into more than 47% of transduced cells (HSCs) at a multiplicity of infection (MOI) of 1000 (a-B in fig. 5). Other tested MOI values (500 and 2000) achieved 37% and 39% integration of eGFP transgene, respectively. Analysis of the same batch of samples by microdroplet digital PCR (ddPCR) confirmed that flow cytometry (FACS) resulted in the result that Bohemine pre-treated and in vitro cultured HSCs showed 40.6% transgene integration after LV-eGFP (MOI 1000) transduction, whereas no positive signal was detected for HSCs not pre-treated Bohemine, and for HSCs receiving LV treatment alone (C-D in FIG. 5). In Bohemine pretreatment HSCs using LV vector transduced ARSA cDNA, multiplicity of infection (MOI) 2000 showed optimal integration efficiency (32.7%; E in fig. 5), whereas MOI 500 showed significantly reduced integration rate (25.8%; p < 0.01). To assess the endogenous transcription of the integrated ARSA cDNA, qPCR primers and probes were designed that bind only to the Bohemine pretreatment group of ARSA mRNA, but not to the endogenous wild-type ARSA mRNA. In this qPCR experiment, it was observed that the expression level of ARSA mRNA was 220-fold higher in Bohemine-treated HSC than in untreated samples of LV-treated cells alone or healthy donors (F in FIG. 5). The data generated confirm that the designed probe did not hybridize to classical ARSA mRNA. Furthermore, since these cells were derived from healthy donors, their ARSA enzyme activity remained unchanged, whether or not subjected to gene editing and correction (G in fig. 5). In addition, to test whether Bohemine + LV vector treatment affects HSC cell differentiation, specific myeloid markers were analyzed. The results show that regardless of the treatment regimen, the proportion of cells expressing CD33 (range 98.6-99.5%), CD11b (37.0-39.7%) or CD66b (23.0-28.1%) was similar (H in FIG. 5), demonstrating that Bohemine +LV vector treatment can increase transgene integration efficiency, thereby maintaining differentiation of HSC cells.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims

1. The application of Bohemine in maintaining the functional characteristics of artificial hematopoietic stem cells in vitro under quiescent conditions, characterized in that, before long-term culture of artificial hematopoietic stem cells, the artificial hematopoietic stem cells are first seeded into a transduction medium and pre-cultured for 2 days with Bohemine added, wherein the working concentration of Bohemine is 200-500 µM; 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;

In the long-term culture of artificial hematopoietic stem cells, StemSpan SFEM medium was used instead of BIT medium, and the artificial hematopoietic stem cells were derived from umbilical cord blood.

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 a transduction medium and pre-cultured for 2 days with the addition of Bohemine, wherein the working concentration of Bohemine is 200-500 µM; 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 mononuclear cells in umbilical cord blood are separated by Ficoll gradient centrifugation, and CD34 ⁺ cells are purified by immunomagnetic separation using a CD34 microbead kit to obtain artificial hematopoietic stem cells.