Rib cartilage-derived stem cells and their combination with multipotent progenitor cells, hematopoietic stem cells or cyclosporin for use in the preparation of a preparation for hematopoietic reconstitution

Abstract

The present application relates to the technical field of hematopoietic reconstitution, and in particular to the application of rib cartilage-derived stem cells and their combination with multipotent progenitor cells, hematopoietic stem cells or cyclosporine in the preparation of a preparation for hematopoietic reconstitution. Rib cartilage-derived stem cells (CD45 ‑ CD51 + ) are derived from rib cartilage and have significant niche restoration capacity. When used alone or in combination with hematopoietic stem cells and multipotent progenitor cells, these cells can enhance hematopoietic function. Rib cartilage-derived stem cells differentiate into important supporting cells that constitute the bone marrow niche, secrete hematopoietic cytokines, and protect hematopoietic stem / progenitor cells. In the treatment of aplastic anemia, rib cartilage-derived stem cells synergize with cyclosporine A to improve immune-mediated bone marrow failure through a dual mechanism. This technical solution solves the technical problem of unsatisfactory effect of hematopoietic stem cell transplantation in the treatment of bone marrow failure and the promotion of hematopoietic reconstitution, and opens up a new way for the treatment of bone marrow failure and hematological diseases.

Description

Technical Field

[0001] This invention relates to the field of hematopoietic reconstitution technology, specifically to the application of costal cartilage-derived stem cells and their combination with pluripotent progenitor cells, hematopoietic stem cells, or cyclosporine in the preparation of formulations for hematopoietic reconstitution. Background Technology

[0002] Bone marrow, as the body's primary hematopoietic organ, plays a crucial role in maintaining vital physiological functions such as oxygen transport, immune defense, and hemostasis through the self-renewal and multi-directional differentiation of hematopoietic stem / progenitor cells (HSPCs). When bone marrow hematopoietic function declines or fails due to genetic defects, immune abnormalities, damage from radiotherapy and chemotherapy, infection, or hematological malignancies, bone marrow failure syndrome can occur. This syndrome manifests as a pancytopenia in peripheral blood, leading to severe anemia, recurrent infections, and bleeding tendencies, seriously threatening the patient's life and health. Against this backdrop, hematopoietic reconstitution has become a core objective in the treatment of bone marrow failure and hematological diseases. Specifically, hematopoietic reconstitution is the process of restoring normal bone marrow hematopoietic function through endogenous repair or exogenous interventions (such as cell transplantation and microenvironment regulation).

[0003] Currently, hematopoietic stem cell transplantation (HSCT) remains the cornerstone therapy for achieving hematopoietic reconstitution and radically curing various hematological malignancies and severe bone marrow failure in clinical practice. It involves infusing hematopoietic stem cells (HSCs) from a healthy donor, allowing them to colonize, proliferate, and differentiate in the recipient's bone marrow, ultimately replacing the damaged hematopoietic system. However, the clinical application and therapeutic efficacy of HSCT are significantly limited by two key factors: firstly, the scarcity of donor HSCs and the limited number of matched donors greatly restrict the widespread adoption of this therapy; secondly, HSCT primarily relies on the functional reconstitution of donor HSCs, but struggles to address potential damage to the recipient's bone marrow microenvironment (i.e., the bone marrow niche) (it cannot address potential bone marrow niche damage). The bone marrow niche, composed of non-hematopoietic cells such as vascular endothelial cells and mesenchymal stem cells, as well as extracellular matrix and signaling molecules, is a crucial site for the survival, colonization, and function of HSCs. Among various pathological conditions (such as long-term radiotherapy and chemotherapy, autoimmune damage, myelofibrosis, etc.), non-hematopoietic cell-mediated abnormalities in bone marrow niche structure and function are often important causes of hematopoietic failure. HSCT cannot specifically repair such damage, leading to delayed or failed hematopoietic reconstitution after transplantation or long-term unstable hematopoietic function in some patients.

[0004] These limitations have prompted researchers to pay increasing attention to the repair mechanisms of the bone marrow microenvironment, and have driven the exploration of novel therapies that directly target damaged bone marrow niches and improve their structure and function, in order to provide more effective treatment strategies for hematopoietic reconstitution. Summary of the Invention

[0005] The present invention aims to provide the application of costal cartilage-derived stem cells in the preparation of formulations for hematopoietic reconstitution or treatment of bone marrow failure, in order to solve the technical problem that the effect of hematopoietic stem cell transplantation in treating bone marrow failure and promoting hematopoietic reconstitution in the prior art is not ideal.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of costal cartilage-derived stem cells in the preparation of formulations for hematopoietic reconstitution or treatment of bone marrow failure.

[0007] The principle behind adopting the above technical solution is as follows: Although costal cartilage-derived stem cells (CDSCs) are primarily used for bone tissue engineering, this study revealed their innovative potential for reconstructing the hematopoietic microenvironment. CDSCs not only differentiate into essential components of the bone marrow niche, such as perivascular cells, endothelial cells, and osteoblasts, but also secrete key hematopoietic cytokines and protect hematopoietic stem / progenitor cells (HSPCs) from radiation-induced damage. This dual mechanism of structural and functional niche restoration represents a substantial advance in hematopoietic restoration strategies.

[0008] The bone marrow (BM) niche plays a crucial role in the maintenance and expansion of hematopoietic stem cells (HSPCs), and BM niche damage affects both hematopoietic and non-hematopoietic cells. CDSCs exhibit significant plasticity, differentiating into multiple cell types. These regenerating cells may promote niche function by producing essential factors such as CXCL12 and SCF. Importantly, CDSCs enhance CFU-F activity and increase the production of various pro-hematopoietic cytokines, including CXCL12, Kit1, Vcam1, Spp1, Adipoq, and Sp7. Studies have shown that CDSCs can effectively regenerate the damaged microenvironment through two key mechanisms: differentiation into functional niche tissue cells and secretion of key hematopoietic cytokines.

[0009] Hematopoietic reconstitution refers to the process of restoring damaged or defective hematopoietic function by transplanting or activating hematopoietic stem cells (HSPCs) in the body, thereby restoring the number and function of various blood cells (red blood cells, white blood cells, platelets, etc.) to normal levels. Its core mechanism is that exogenous or endogenous HSPCs colonize, self-renew, and differentiate into various mature blood cells in a suitable bone marrow microenvironment (bone marrow niche), gradually rebuilding a complete hematopoietic and immune system. Hematopoietic stem cell transplantation (HSCT) is a specific application scenario.

[0010] Bone marrow failure is a syndrome caused by decreased or failed function of the bone marrow's hematopoietic tissue, leading to a reduction in all blood cells (red blood cells, white blood cells, and platelets) in the peripheral blood. Symptoms include anemia, increased susceptibility to infection, and bleeding tendency. Essentially, it is caused by a decrease in the number or functional defects of hematopoietic stem cells (HSPCs), or an abnormal bone marrow microenvironment, resulting in an inability of hematopoiesis to meet the body's needs. If bone marrow failure is not intervened in time, it may progress to severe aplastic anemia, myelodysplastic syndrome (MDS), or even leukemia, requiring treatment such as immunosuppressive therapy and hematopoietic stem cell transplantation to rebuild hematopoietic function.

[0011] The bone marrow (BM) niche is a complex microenvironment crucial for maintaining hematopoietic stem cells (HSCs) and sustaining lifelong blood cell production. Composed of hematopoietic and supporting stromal cells, including perivascular cells, endothelial cells, osteoblasts, and mesenchymal stem cells (MSCs), the BM niche precisely regulates HSC self-renewal, differentiation, and stress responses through complex cell interactions. However, this fragile system is particularly vulnerable to damage from chemotherapy, radiation therapy, immune dysregulation, and age-related degeneration, often leading to severe hematopoietic impairment.

[0012] Furthermore, the costal cartilage-derived stem cells are derived from costal cartilage and have the surface marker CD45. - CD51 + . cells.

[0013] Furthermore, the costal cartilage-derived stem cells are prepared by the following method: costal cartilage is digested and dissociated using type II collagenase, and then resuspended in phosphate buffer containing fetal bovine serum to obtain an enzymatically separated cell collection; then, cells that do not express CD45 but express CD51 on the surface are screened by flow cytometry to obtain costal cartilage-derived stem cells.

[0014] Furthermore, the formulation includes costal cartilage-derived stem cells and other stem cells; the other stem cells are hematopoietic stem cells and / or pluripotent progenitor cells.

[0015] The technical principle behind the above solution is as follows: This technical solution utilizes costal cartilage-derived stem cells in combination with one or both of hematopoietic stem cells (HSCs) and pluripotent progenitor cells (MPPs) to effectively promote hematopoietic reconstitution and treat bone marrow failure.

[0016] CDSCs effectively promote hematopoietic remodeling with minimal support from HSCs. Although MPPs alone cannot maintain long-term engraftment due to their limited self-renewal capacity, their combination with CDSCs significantly improves survival rates. Typically, transplantation requires at least 5 × 10⁶ CDSCs. 5Each BM cell line contains approximately 300 HSCs to ensure adequate hematopoietic reconstitution. In stark contrast, this technique, based on CDSCs, achieves comparable hematopoietic function with fewer than 10 HSCs, representing a significant reduction in the number of HSCs required. Comprehensive analysis of hematopoietic progenitor cell populations, mature immune cell reconstitution, and organ recovery confirms that CDSC-supported transplantation performance is comparable to bone marrow transplantation (BMT).

[0017] Compared to mesenchymal stem cells (MSCs), CDSCs exhibited significantly enhanced niche restoration capabilities. MSCs are thought to promote hematopoietic recovery by repairing damaged BM niches and secreting hematopoietic cytokines. In this protocol, co-transplantation of CDSCs and MPPs significantly improved the survival rate of experimental animals, while co-transplantation of MSCs and MPPs failed to rescue mice. These findings suggest that CDSCs have a stronger BM niche restoration capacity than MSCs. CDSCs are easier to expand in vitro and are less prone to functional loss during in vitro culture; therefore, CDSCs show greater potential as seed cells for BM niche restoration.

[0018] Furthermore, every 5×10 5 Each costal cartilage-derived stem cell corresponds to ≥50 hematopoietic stem cells, with each 5 × 10 5 Each costal cartilage-derived stem cell corresponds to ≥1000 pluripotent progenitor cells. Preferably, the cell ratio of costal cartilage-derived stem cells to hematopoietic stem cells is 5 × 10⁻⁶. 5 The survival rate of experimental animals reached 80-88% (dual transplantation of CDSCs and HSCs into mice irradiated with a lethal dose); the cell ratio of costal cartilage-derived stem cells to pluripotent progenitor cells was 5×10⁻⁵. 5 :1000, the survival rate of experimental animals can reach 55-70% (CDSCs and MPPs combined transplantation lethal dose irradiated mice).

[0019] Furthermore, the bone marrow failure mentioned is bone marrow failure caused by radiation damage.

[0020] Moderate to high doses of whole-body radiation can impair the function of hematopoietic stem cells (HSCs), leading to acute or long-term myelosuppression. In our protocol, a complete myeloablative radiation dose of 9.5 Gy was administered to eradicate endogenous HSCs; however, the experimental animals maintained functional hematopoiesis under the influence of CDSCs and with a low number of donor HSCs. This suggests that CDSCs may protect residual host HSCs from radiation-induced damage, and the experimental data also support this view: reduced apoptosis and DNA damage in HSCs and MPPs. Furthermore, these protected HSCs retained their functional potential, demonstrating long-term engraftment capacity in secondary transplantation and competitive reproliferation assays.

[0021] This technical solution also provides the application of a composition formed from costal cartilage-derived stem cells and cyclosporine A in the preparation of a drug for treating aplastic anemia.

[0022] Furthermore, the ratio of the costal cartilage-derived stem cells to cyclosporine A is 5 × 10⁻⁶. 5 Quantity: 5mg.

[0023] Aplastic anemia is a clinically challenging syndrome of bone marrow failure characterized by pancytopenia and bone marrow hypoplasia, resulting from hematopoietic stem cell (HSC) depletion and microenvironment dysfunction. Current treatment options, including hematopoietic stem cell transplantation and immunosuppressive therapy, are limited by patient age, physical condition, and potential side effects. Although cyclosporine A (CsA) is often used in combination with anti-thymocyte globulin (ATG) for the treatment of aplastic anemia, CsA monotherapy has shown limited efficacy and is associated with significant adverse reactions. CDSCs can be readily cultured and expanded in vitro and can promote hematopoietic reconstitution, making them a promising alternative for therapeutic applications. While CDSCs cultured alone only moderately prolong the survival of mice with aplastic anemia, the combination of CDSCs and CsA significantly improved therapeutic efficacy. This combination enhanced BM cell counts, reduced apoptosis rates, and inhibited CD4+ in BM, peripheral blood, and spleen. + and CD8 + T-cell infiltration and accelerated blood cell recovery. Therefore, the combination of CDSCs and CsA shows therapeutic potential comparable to other established treatments for aplastic anemia.

[0024] This technical solution also provides the application of costal cartilage-derived stem cells in the preparation of formulations that promote the hematopoietic reconstitution function of hematopoietic stem cells.

[0025] Furthermore, costal cartilage-derived stem cells are used to reduce the number of hematopoietic stem cells or pluripotent progenitor cells in transplantation for hematopoietic reconstitution; the costal cartilage-derived stem cells are derived from costal cartilage and have the surface marker CD45. -CD51 + . cells.

[0026] Furthermore, costal cartilage-derived stem cells are used to increase the number and activity of hematopoietic stem cells.

[0027] This technical solution also provides a method for preparing costal cartilage-derived stem cells with the function of promoting hematopoietic reconstitution of hematopoietic stem cells and / or pluripotent progenitor cells. The method involves digesting and dissociating costal cartilage with type II collagenase, then resuspending it in phosphate buffer containing fetal bovine serum to obtain an enzymatically separated cell collection; then, cells that do not express CD45 but express CD51 on the surface are screened by flow cytometry to obtain costal cartilage-derived stem cells.

[0028] Furthermore, the costal cartilage was completely immersed in DMEM / F12 medium containing 0.2% type II collagenase and digested at 90 rpm for 20 minutes. Then, the costal cartilage was washed with phosphate-buffered saline (PBS). The costal cartilage was then completely immersed in DMEM / F12 medium containing 0.2% type II collagenase and digested at 90 rpm for 35 minutes. The costal cartilage could be completely dissociated. The dissociated cells were resuspended in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) to obtain enzymatically isolated costal cartilage-derived stem cells, i.e., the enzymatically isolated cell collection.

[0029] The technical principle of this technical solution is as follows: The technical principle of this approach is based on the ability of CDSCs to repair the BM niche and their synergistic regulatory effect on hematopoietic stem / progenitor cell function. CDSCs repair the BM niche through multiple pathways and achieve hematopoietic reconstitution and treatment of bone marrow failure: (1) Structural and functional restoration of the BM niche: CDSCs (surface marker is CD45) - CD51 + They can differentiate into key supporting stromal cells in the BM niche, including bone marrow mesenchymal stem cells, endothelial cells, and osteoblasts, directly replenishing the loss of cellular components caused by radiation, immune damage, etc., and rebuilding the structural integrity of the niche. At the same time, CDSCs secrete hematopoietic cytokines (such as stem cell factor, vascular endothelial growth factor, etc.), activate the endogenous repair pathways of damaged niches, and restore their supporting function for HSCs, including regulating the balance between HSC resting and proliferation, and promoting their colonization and differentiation.

[0030] When CDSCs are co-transplanted with MPPs or HSCs in a specific ratio, the hematopoietic reconstitution effect can be achieved with traditional bone marrow transplantation (BMT) by repairing the BM niche and enhancing hematopoietic cell function, while reducing the amount of HSCs / MPPs used, effectively alleviating the problem of donor HSCs scarcity.

[0031] (2) Protection and functional enhancement of hematopoietic stem / progenitor cells: CDSCs can inhibit radiation-induced apoptosis of hematopoietic stem / progenitor cells and the accumulation of DNA damage, helping residual hematopoietic stem / progenitor cells to establish new niches. In addition, CDSCs can enhance the colonization efficiency and proliferation activity of HSCs by improving the niche microenvironment and direct intercellular interactions. When co-transplanted with MPPs or HSCs, CDSCs can significantly improve the speed and stability of hematopoietic reconstitution.

[0032] (3) Synergistic therapeutic effect: In the aplastic anemia model, CDSCs and CsA work synergistically: CsA mainly inhibits the attack of abnormal immune system on hematopoietic cells and niches, while CDSCs focus on repairing damaged BM niches. Through the dual mechanism of "immune regulation + microenvironment repair", the two can specifically improve immune-mediated bone marrow failure.

[0033] In summary, CDSCs, as a type of multifunctional stem cell that combines BM niche repair, hematopoietic cell protection, and synergistic regulation, provide a novel strategy for hematopoietic reconstitution and bone marrow failure treatment by targeting and repairing damaged BM niches. They have significant advantages, especially in addressing the shortcomings of donor scarcity and the inability to repair BM niche damage in traditional hematopoietic stem cell transplantation (HSCT).

[0034] The beneficial effects of this technical solution are as follows: (1) Repairing the BM ecological niche and overcoming the limitations of traditional transplantation CD45 - CD51 + Phenotypic CDSCs can differentiate into crucial supporting cells that constitute the bone marrow niche, repairing structures damaged by radiation, chemotherapy, or immune responses. Their secreted hematopoietic cytokines activate intrinsic repair pathways, restoring the bone marrow niche's support function for hematopoietic stem cells. This unique mechanism overcomes the limitation of traditional hematopoietic stem cell transplantation (HSCT) in repairing damaged bone marrow niches, fundamentally reducing the risk of delayed or failed hematopoietic reconstitution.

[0035] (2) Reduce the amount of hematopoietic cells used and alleviate the limitation of donor resources. Traditional bone marrow transplantation requires a large number of hematopoietic stem / progenitor cells to ensure the success rate of hematopoietic reconstitution. However, when CDSCs are used in combination with MPPs or hematopoietic stem cells in a specific ratio, only a small number of hematopoietic stem cells are needed to achieve the same effect as traditional transplantation, greatly reducing the demand for donor HSPCs.

[0036] (3) Synergistic effect of cyclosporine A to optimize the treatment of aplastic anemia In an aplastic anemia model, CDSCs and cyclosporine A (CsA) worked synergistically to significantly improve the survival rate of experimental animals. Compared with CsA alone, this combination reduced dependence on immunosuppressants and related side effects, providing a more effective treatment for immune-mediated bone marrow failure.

[0037] (4) It is superior to mesenchymal stem cells and has ideal clinical application potential. Compared to mesenchymal stem cells (MSCs), CDSCs exhibit stronger bone marrow niche repair capabilities. Furthermore, due to their lower difficulty in in vitro expansion and high functional stability during culture, CDSCs are more suitable for standardized preparation as seed cells, promoting their translation to clinical applications.

[0038] In summary, CDSCs effectively address the issues of donor scarcity and insufficient bone marrow niche repair faced by traditional HSCT through multiple mechanisms, significantly improving the efficiency, stability, and applicability of hematopoietic reconstitution, and opening up new avenues for the treatment of bone marrow failure and hematological diseases. Attached Figure Description

[0039] Figure 1The experimental results of isoCDSCs and BM in rescuing lethal radiation-exposed mice in Example 1 are as follows: (A. Equation diagram of isoCDSCs-T generation process; B. Survival curves of mice after isoCDSCs transplantation into isoCDSCsT and bone marrow transplantation into BMT; C. Lineage distribution of donor-derived cells (CD45.1 / ZsGreen) in peripheral blood (PB) of isoCDSCs-T (n=7) and BMT (n=6) at 8 weeks post-transplantation; DE. Total cell count and organ index of spleen and thymus in recipient mice (isoCDSCs-T, n=8) and healthy controls (n=8) at 16 weeks post-transplantation; F. Comparison of PB chimerism levels between isoCDSCs-T (n=41) and BMT (n=16) at 8 weeks post-transplantation; G. Comparison of PB chimerism levels between isoCDSCs-T (n=12) and BMT (n=8) at specified time points post-transplantation; HI. For isoCDSCs-T (n=12) and BMT (n=8), the percentage of HSPCs and donor chimerism in BM at 16 weeks post-transplantation; J. An overview of non-competitive transplantation of donor-derived BM cells (donor-HSCT / CD45.1) and irradiated BM cell rescue (rescue-HSCT / CD45.2) from the primary recipient of isoCDSCs-T at 16 weeks post-transplantation; K. Survival of recipients of non-competitive transplantation of donor-HSCT and rescue-HSCT; L. Donor chimerism in BM of recipients of non-competitive secondary transplantation of donor-HSCT (n=8) and rescue-HSCT (n=4) at 16 weeks post-transplantation; M. Donor chimerism in PB of recipients of non-competitive transplantation of donor-HSCT (n=4) and rescue-HSCT (n=5) at the specified time point post-transplantation; N. Lineage distribution of donor-derived cells (CD45.1 / CD45.2) in non-competitive secondary transplant recipients (PB): donor-HSCT (n=4 at 2 months; n=8 at 4 months) and salvage-HSCT (n=4 at 2 months; n=4 at 4 months); *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant).

[0040] Figure 2 The experimental results of co-transplantation of CDSCs with HSCs and MPPs in Example 1 successfully rescued lethally irradiated mice (A. Compositional analysis of isoCDSCs revealed CD45). - CD51 + The main population of CDSCs (left panel) and a small number of HSCs / MPPs (middle panel) are shown in the right panel, which quantitatively shows the number of HSCs / MPPs per transplanted mouse (5 × 10⁻⁶). 5A. Overview of the experimental procedures for HSCs and MPPs transplanted alone or in combination with CDSCs; B. Survival rate after transplantation of HSCs and CDSCs alone or in combination; C. Survival rate after transplantation of MPPs alone or in combination with CDSCs or MSCs; D. Donor chimerism level in peripheral blood PB 4 weeks after transplantation of HSCs alone (n=6), HSCs+CDSCs (n=8), and MPPs+CDSCs (n=5); F. Percentage of HSCs in BM 8 weeks after transplantation of HSCs alone (n=3), HSCs+CDSCs (n=3), and MPPs+CDSCs (n=4); G. Total cell count and organ index in BM, spleen, and thymus 8 weeks after transplantation of HSCs and MPPs alone or in combination with CDSCs; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0041] Figure 3 The results of the study on the effect of CDSCs in restoring the damaged BM niche to promote hematopoiesis in Example 1 are as follows: (A. Experimental procedure for isolating BMSCs from irradiated tibias and measuring CFU-F at 3 weeks post-transplantation; B. C. Images of CFU-F measurement results of colony-forming unit fibroblasts in BMSCs from the CDSCs group, BMT group, control group, and healthy group at 3-6 weeks post-transplantation; D. Experimental procedure for sorting individual HSCs from CDSCs, BMT (2-3 weeks post-transplantation), control group, and healthy group; E. Clonal size of 100 sorted individual HSCs after 14 days of methylcellulose culture; F. F. Percentage of HSCs (including LSKs, LT-HSCs, ST-HSCs, and MPPs) in CDSCs (n=8), BMT (n=5), control group (n=5), and healthy group (n=6); *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0042] Figure 4The results of the study on the effect of CDSCs in regenerating BM endothelial and stromal cells in the damaged BM niche of Example 1 are as follows: (A. Schematic diagram of transplantation of purified CDSCs and BM into irradiated mouse tibias; B. Flow cytometry diagram illustrating BMSCs differentiated from CDSCs; C. Donor chimerism in BMSCs of irradiated tibias 3-6 weeks after transplantation of purified CDSCs (n=17) and BMTs (n=6); D. Quantitative detection results of differentiated BMSCs in irradiated tibias of purified CDSCs (n=13), BMTs (n=6), control group (n=7) and healthy group (n=11); *: P<0.05, **: P<0.01.)

[0043] Figure 5 The study of the effect of cultured CDSCs on promoting hematopoietic recovery in Example 1 included: (A) Hematological parameters (RBC, PLT, WBC) of cultured CDSCs (n=9), BMT (n=5), control group (n=9), and healthy group (n=5) 1-4 weeks after transplantation; (B) Survival rate of mice in femoral transplantation experiments with different transplantation methods in lethal irradiated mice; (C) Donor chimerism of HSCs (50) or MPPs (1000) transplanted alone or co-transplanted with cultured CDSCs in peripheral blood PB of recipient mice 4 weeks after transplantation: HSCs (n=4), HSCs + cultured CDSCs (n=5), MPPs + cultured CDSCs (n=7); (D) Apoptosis level of BM Lin- cells 1 week after transplantation of cultured CDSCs; (H) DNA damage level in BM MPPs 3 weeks after transplantation of cultured CDSCs; (F) UMAP cluster analysis of primary and cultured CDSCs; (H) Expression of genes related to BMSCs, endothelial cells, and chondrocytes in primary and cultured CDSCs; L. Developmental and differentiation trajectories of primary and cultured CDSCs; M. Pseudo-temporal analysis of genes related to BMSCs, endothelial cells, and chondrocytes in primary and cultured CDSCs; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.).

[0044] Figure 6The experimental results of CDSCs combined with CsA in treating aplastic anemia in Example 2 are shown below: (A. Schematic diagram of the treatment process using cultured CDSCs and CsA combination in aplastic anemia (AA) mice; B. Survival curves of AA group, control group, CsA alone group, and CsA+CDSCs group; C. Hematological parameters (RBC, PLT) and total BM cell count 12-16 days after AA induction; Gray, black, red, and blue represent control group (n=5), AA group (n=5), AA+CsA+CDSCs group (n=5), and AA+CsA group (n=5), respectively; F. CD4 counts in BM, PB, and spleen of AA, control group, CsA alone group, and CsA+CDSCs group 12-16 days after transplantation.) + and CD8 + Percentage of T cells; J. Flow cytometry plots and BM cells (excluding CD4+) in AA (n=3), control (n=3), CsA alone (n=3), and CsA+CDSCs (n=3) groups 12–16 days after establishment of the AA mouse model. + and CD8 + Percentage of T cell apoptosis; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant).

[0045] Figure 7 Example 3 shows the gene expression characteristics and cell proliferation of CDSCs from different sources (A. UMAP diagrams of human and mouse CDSCs; B. Characteristic gene expression of human and mouse CDSCs; C. Correlation analysis of gene expression in human and mouse CDSCs; D. Cell morphology of human and mouse CDSCs during in vitro culture; E. Cell growth of human and mouse CDSCs during in vitro culture, including P6 and P1 generations).

[0046] Figure 8 Example 3 shows the trilineage differentiation of human CDSCs and the expression of BMSCs markers in cultured human CDSCs (A. In vitro differentiation of human CDSCs into bone (Alizarin Red staining), cartilage (Alcian Blue staining), adipose tissue (Oil Red O staining), fibroblasts (COL1A1 immunofluorescence staining), and myoblasts (white light); B. Flow cytometry images showing the expression of BMSCs markers in cultured human CDSCs; C. Flow cytometry images showing the expression of BMSCs markers in cultured mouse CDSCs). Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0048] Example 1: Study on CDSCs to rescue bone marrow hematopoietic failure (1) Cell origin Costal cartilage-derived stem cells (CDSCs) were obtained by the following method: CDSCs were isolated from the costal cartilage of newborn mice (born within one week) of the CD45.1 or ZsGreen C57BL / 6 strain (Cyagen, Guangzhou, China). The isolation process involved enzymatic digestion and dissociation. Enzymatic digestion was performed in two consecutive steps: the first step aimed to remove the muscle tissue surrounding the costal cartilage, while the second step focused on isolating CDSCs from the purified costal cartilage. The enzyme solution consisted of 0.2% type II collagenase (C6885-1G, Sigma-Aldrich, USA) dissolved in DMEM / F12 (Cytiva, SH30023.01). First, the costal cartilage was digested at 90 rpm for 20 minutes and then washed with PBS (Gibco, C10010500BT). Subsequently, a second enzymatic digestion was performed at 90 rpm for 35 minutes. Finally, the CDSCs were mechanically dissociated and resuspended in PBS containing 2% FBS (fetal bovine serum). The cells obtained in this step were named enzymatically isolated CDSCs (isoCDSCs). CD45 was isolated by flow cytometry. - CD51 + Cells (i.e., cells that do not express the hematopoietic marker CD45 on their surface but express integrin αV CD51) were used to obtain purified CDSCs, which were named CDSCs. In this protocol, cells were harvested by enzymatic digestion of neonatal mouse costal cartilage; these cells were also referred to as enzymatically isolated CDSCs (isoCDSCs). The purified CDSCs were seeded into minimum essential medium (HyClone, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Thermo Scientific, USA) and cultured in 6 cm cell culture dishes. Cells were cultured at 37°C in a 5% CO2 incubator, with the medium refreshed 2-3 times per week and passaged every 5 days. CDSCs are derived from costal cartilage, with CD45 as the surface marker. - CD51 + . cells.

[0049] (2) Experimental methods (2.1) Irradiation and transplantation experiments Mice were irradiated with a lethal dose to induce bone marrow hematopoietic failure. Recipient mice (16 weeks old, CD45.2 mice, Vital River Laboratory Animal Technology Co., Ltd., Chongqing Tengxin Biotechnology Co., Ltd.) received 9.5 Gy irradiation one day prior to transplantation, specifically administered in fractionated doses of 5 and 4.5 Gy at 4-hour intervals using Co-60 γ-rays. The irradiated mice then underwent CDSCs transplantation (isoCDSCs-T) and BM cell transplantation (BMT). In isoCDSCs-T, the aforementioned irradiated mice received an intraorbital intravenous injection of 5 × 10⁵ gamma rays. 5 isoCDSCs were isolated using the method described above. In BMT, 5 × 10⁶ cells were injected intraorbitally. 5 Bone marrow cells (BMs) were obtained from the bone marrow tissue of newborn or 8-12 week old CD45.1 or ZsGreen mice. BM cells were flushed from the bone marrow tissue using a 1 ml syringe, resuspended in PBS buffer containing 2% serum, impurities were removed using a 70 μm cell filter, red blood cells were removed using red blood cell lysis buffer, and the cells were washed once with buffer to obtain experimental BM cells. BM cells refer to all types of cells present in the bone marrow, including hematopoietic cells, stromal cells, and other supporting cells, which together maintain the hematopoietic function and microenvironmental stability of the bone marrow.

[0050] In addition, HSC transplantation (HSCT), MPP transplantation (MPPT), and combined transplantation of multiple cell types were also performed. HSCs (derived from ZsGreen mice, Lin was isolated from BM cells) - Sca1 + cKit + CD48 - Cells), MPPs (derived from ZsGreen mice, Lin isolated from BM cells) - Sca1 + cKit + CD48 + CD150 - Cells were injected intravenously into irradiated recipient mice at doses of 50 or 1000 cells, respectively, or in combination with 5 × 10⁻⁶ cells. 5 One CDSC (derived from CD45.1 mice) was injected intraorbitally into irradiated recipient mice. The irradiated recipient mice were 8-12 week old wild-type recipient mice (CD45.2) subjected to lethal irradiation, as described above. For the non-competitive transplantation experiment of isoCDSCs-T, 5 × 10⁶ CDSCs were injected intraorbitally into irradiated recipient mice. 5 One rescued BM cell (rescued HSCT / CD45.2) was transplanted into lethally irradiated 8-12 week old CD45.1 recipient mice via intraorbital intravenous injection, while 5 × 105 Donor-derived BM cells (donor HSCT / CD45.1) were transplanted into lethally irradiated 8-12 week old CD45.2 recipient mice via intraorbital vein injection. For sublethal irradiation and transplantation assays, 5 × 10⁵ cells of P2 or P3 passaged cells were injected bilaterally into the medullary canal of the femur. 5 One CDSC was transplanted into 8-12 week old wild-type recipient mice (CD45.2) subjected to sublethal irradiation (5 Gy, Co-60 γ-rays). The study on the hematopoietic-promoting effect of cultured CDSCs (cells passaged from P2 or P3) was conducted as follows: 8-12 week old mice were subjected to the aforementioned lethal irradiation, and then 50 HSCs or 1000 MPPs were transplanted individually into the irradiated mice, or HSCs and MPPs were co-transplanted with cultured CDSCs (5 × 10⁻⁶ cells). 5 ) transplanted into irradiated mice, or HSCs and MPPs combined with MSCs (5×10) 5 (Number) HSCs and MPPs were transplanted into irradiated mice. Cultured CDSCs and MSCs were transplanted via intraorbital vein injection, while cultured CDSCs and MSCs were transplanted via bilateral intramedullary injection. For single-irradiation tibial assays, the tibia of mice was irradiated with 12 Gy the day before transplantation. Then, purified 5 × 10⁶ HSCs and MPPs were injected intramedullary. 5 One CDSC (ZsGreen) was transplanted into the irradiated tibia of 8-12 week old wild-type recipient mice (CD45.2).

[0051] (2.2) CFU-F determination and CFU determination Washed BM cells, primarily composed of hematopoietic cells, were removed from mice irradiated with a single tibia. The remaining tibia was then continuously digested with enzymes at 110 rpm for 30 minutes. The enzyme solution was DMEM containing 1 mg / mL type IV collagenase (17104019, Sigma-Aldrich, USA) and 0.5 mg / mL hydrolase (D4693-1G, GIBCO, USA). After digestion, cells were harvested by repeated pipetting and washing, and then resuspended in PBS buffer containing 2% FBS. Finally, CD45 cells were sorted from the digested cell suspension using a fine-cell sorting system. - CD31 - Ter119 - BMSCs.

[0052] CFU-F assay: 20,000 BMSCs were seeded into 6-well plates, and 60,000 BMSCs were seeded into 6 cm cell culture dishes. The cell culture medium consisted of a MesenCult amplification kit (05513, Stem Cell Technologies, Canada) and essential medium (supplemented with 10% FBS and 1% penicillin / streptomycin), and was refreshed every 5 days. After 10 days of culture, the wells were washed twice with PBS and stained with crystal violet staining solution (C0121, Beyotime Biotechnology, China) for 10 minutes at room temperature, followed by three additional washes with PBS. Finally, the stained cell colonies were quantified and imaged.

[0053] CFU assay: 96-well U-bottom plates were coated with 100 μL of methylcellulose medium (M3434, Stem Cell Technologies, Canada), supplemented with 1% penicillin / streptomycin, and incubated at 4°C for subsequent experiments. Subsequently, single HSCs from donor-derived HSCs and rescued HSCs from isoCDSCs-T transplant recipients were added to the pre-coated 96-well plates and cultured at 37°C in a 5% CO2 incubator. After 14 days of culture, cell colonies were quantified and measured using an inverted fluorescence microscope.

[0054] (3) Experimental results (3.1) isoCDSCs-T can effectively salvage bone marrow hematopoietic failure, and its effect is comparable to that of bone marrow transplantation. The procedure for transplanting isoCDSCs into lethally irradiated mice is as follows: Figure 1 As shown in Figure A. Transplantation of 5×10⁶ cells / year. 5 The survival rate of isoCDSCs (isoCDSCs-T) was 84%, comparable to that of BMT. Figure 1 B). isoCDSCs-T and BMT showed similar lymphatic and bone marrow lineage remodeling in peripheral blood (B). Figure 1 C). Furthermore, recipients of isoCDSCs-T showed total cell counts and organ indices in the BM, thymus, and spleen comparable to those in age-matched healthy mice. Figure 1 DE. Similar numbers and proportions of hematopoietic stem / progenitor cells (HSPCs) were observed in the isoCDSCs-T group and the BMT group. Figure 1 H). Analysis of the proportions of mature blood cells, T and B cells in lymph nodes and thymus in BM, as well as the number of T cells, B cells, NK cells and granulocytes in peripheral blood and spleen, showed no significant difference between isoCDSCs-T and BMT. To assess long-term multi-lineage reconstitution capacity, a second non-competitive transplantation experiment was conducted ( Figure 1J). Both salvage HSCTs derived from isoCDSCs-T receptors and donor-derived HSCTs showed a survival advantage. Figure 1 K). Although chimerism is higher in peripheral blood for donor-derived HSCT, no significant difference was observed in BM ( Figure 1 LM). Donor-derived HSCT exhibits classic multilineage hematopoietic reconstitution features, while salvage HSCT shows T-lymphocyte-biased reconstitution two months post-transplantation, transitioning to myeloid-biased reconstitution at four months. Figure 1 N). Furthermore, at 16 weeks post-secondary transplantation, salvaged HSCTs and donor-derived HSCTs showed comparable levels of chimerism and cell counts (excluding granulocytes). Overall, isoCDSCs-T achieved hematopoietic reconstitution efficacy comparable to BMT, except for a slight difference in chimerism rate.

[0055] (3.2) IsoCDSCs-T showed better efficacy than BMT in reconstructing the BM niche. To further investigate the potential mechanisms of isoCDSCs-T action comparable to BMT, RNA sequencing analysis was performed on BM samples from BMT and isoCDSCs-T recipients. Principal component analysis (PCA) revealed distinct clusters in three groups: healthy controls, BMT recipients, and isoCDSCs-T recipients. GO term enrichment analysis showed that genes highly enriched in the isoCDSCs-T group were associated with extracellular structural organization, matrix remodeling, collagen metabolism, and cartilage and bone development. In contrast, genes highly enriched in the BMT group were primarily involved in the regulation of B cell and lymphocyte activation, as well as B cell-mediated immunity. Overall, these GO analysis results suggest that the isoCDSCs-T group may exhibit a greater tendency to repair the BM niche of radiation damage compared to the BMT group.

[0056] The genetic profiles of the three groups were examined for correlations with the top 800 genes associated with various bone marrow mesenchymal stem cells (BMSCs), osteoblasts, and vascular endothelial cells. Analysis showed that the isoCDSCs-T group exhibited a higher correlation with these BM niche-related cell types compared to the other two groups. Furthermore, the isoCDSCs-T group showed significantly higher expression levels of several pro-hematopoietic factors, such as CXCL12, Kitl (SCF), Vcam1, Spp1, Adipoq, and Sp7, which have previously been reported to promote hematopoiesis. Immunofluorescence staining further confirmed that isoCDSCs-T expressed characteristic markers of endothelial cells (endomucin), perivascular cells (CXCL12 and Lepr), and osteoblasts (Sp7). In summary, these findings suggest that isoCDSCs-T may participate in hematopoietic remodeling by promoting the remodeling of the BM niche in radiation-damaged cells.

[0057] (3.3) Co-transplantation of CDSCs promotes hematopoietic reconstitution by enhancing the bone marrow niche. To investigate the cellular mechanisms by which isoCDSCs-T cells affect hematopoietic reconstitution, the cellular composition of isoCDSCs was analyzed in detail. Flow cytometry showed that the transplanted cell population was mainly composed of CD45 cells. - CD51 + CDSCs (referred to as CDSCs) are composed of components with minimal contributions from HSCs and MPPs. Specifically, at 5×10 5 Of the CDSCs, only 5.65 were HSCs and 24.35 were MPPs. Figure 2 A). Existing BM cell transplantation (BMT) techniques typically require a certain level of HSCs content in BM cells, typically 5 × 10⁻⁶. 5 To ensure effective hematopoietic reconstitution, each BM cell needs to contain at least 300 HSCs. For example, in the BMT experiment conducted in this technical protocol, the number of HSCs in the BM cells was ≥300. The experimental results in (3.1) and (3.2) show that although the number of HSCs and MPPs in isoCDSCs-T was less than 300, isoCDSCs-T and BMT achieved similar hematopoietic reconstitution effects. This indicates that using CDSCs in combination with HSCs and MPPs for hematopoietic reconstitution can significantly reduce the demand for HSCs. CDSCs can effectively promote hematopoietic reconstitution with minimal HSC support. CDSCs effectively solve the problem of HSC scarcity faced by traditional HSCT, and the in vitro culture method for CDSCs is simple and easy to implement, further improving the efficiency, stability, and applicability of hematopoietic reconstitution.

[0058] To assess the potential of CDSCs to restore the BM niche during hematopoietic remodeling after radiation injury, 5×10 5 CDSCs (CD45) sorted by flow cytometry - CD51 + CDSCs) combined with 50 purified HSCs or 1,000 MPPs were transplanted into lethally irradiated mice. Figure 2 B). The results showed that neither CDSCs nor MPPs alone could save lethally irradiated mice, while a specific number of HSCs alone saved 60% of the irradiated mice (B). Figure 2 CD). Notably, the combination of CDSCs and HSCs rescued 88% of irradiated mice (CD). Figure 2 C). Co-transplantation of CDSCs and MPPs also saved 70% of irradiated mice ( Figure 2 (D) This indicates that CDSCs may have the ability to revitalize damaged BM niches, thus providing an optimal hematopoietic microenvironment for MPPs, which themselves lack long-term hematopoietic remodeling potential. Furthermore, 5×10 5 Effective hematopoietic reconstitution can be achieved by combining 1 CDSC with 50 purified HSCs, while existing BMT techniques require 5 × 10 5 Each BM cell needs to contain at least 300 HSCs. This data comparison also demonstrates the advantage of CDSCs over BM cells in hematopoietic remodeling.

[0059] Given previous reports that mesenchymal stem cells (MSCs) can aid in the recovery of damaged bone marrow microenvironments, we further investigated the co-transplantation of MSCs and MPPs. The transplantation method was the same as for co-transplantation of CDSCs and MPPs, using an equal volume of MSCs instead of CDSCs. Specifically, the MSCs were bone marrow-derived mesenchymal stem cells (BMSCs,® Oricell, MUBMX-01001, Cyagen, China), characterized by microcytology and demonstrating high expression levels (>70%) of CD29, CD44, and sca-1. However, this method failed to rescue any of the experimental mice ( Figure 2 D), indicating that CDSCs exhibit superior BM niche maintenance capacity compared to BMSCs. Peripheral blood chimerism analysis showed that CDSCs+MPPs transplantation resulted in a low level of chimerism similar to that observed in the isoCDSCs-T group (D). Figure 2 E). Furthermore, analysis of the proportion of HSCs showed that CDSCs+MPPs exhibited hematopoietic reconstitution efficacy comparable to the HSCs group (Figure 3F). Similarly, CDSCs+MPPs showed consistency with HSCs in organ indices and total cell counts in the BM, thymus, and spleen (E). Figure 2 (G and H). In summary, these findings suggest that CDSCs may promote hematopoietic reconstitution by enhancing the bone marrow microenvironment. Experimental data show that co-transplantation of CDSCs and MPPs is far more effective than co-transplantation of MSCs and MPPs. The inventors believe this may be because CDSCs can differentiate into vascular endothelial cells and BMSCs, participating in the reconstitution of the bone marrow microenvironment. Bone marrow mesenchymal stem cells (BMSCs) are enriched in the perivascular microenvironment of the bone marrow and interact closely with endothelial cells and HSCs, thereby modulating the bone marrow microenvironment and promoting hematopoietic reconstitution. Therefore, BMSCs are mesenchymal cells commonly used in existing technologies in combination with HSCs for hematopoietic reconstitution and bone marrow failure treatment. This experiment demonstrates that CDSCs exhibit a more ideal hematopoietic reconstitution function than commonly used BMSCs in existing technologies, achieving unexpected technical results.

[0060] (3.4) Study on the effects of CDSCs on damaged BM niche CDSCs revitalize damaged BM niches to promote hematopoiesis. Irradiation permanently damages the bone marrow stromal microenvironment, which is difficult to repair through circulation. To investigate the effects of CDSCs on the irradiated BM niche and the recovery of hematopoietic function, colony-forming unit fibroblast (CFU-F) assays were performed, demonstrating that CDSC transplantation significantly enhanced the proliferation of total bone marrow mesenchymal stem cells (BMSCs) compared to controls and BMT. Figure 3 AC). This enhanced CFU-F activity promotes HSC function, such as increased large colony formation and reduced colony formation failure in single HSC CFU assays. Figure 3 DE). Furthermore, compared to the control, CDSCs increased Lin. - SCA1 + cKit + The percentage and number of LSKs (a population of early hematopoietic stem / progenitor cells with high hematopoietic potential in the bone marrow, playing a central role in the maintenance and regeneration of the hematopoietic system) and long-term hematopoietic stem cells (LT-HSCs) Figure 3 FG), and the percentage relative to control MPPs ( Figure 3 I). However, no significant differences were observed in the percentage and number of short-term hematopoietic stem cells (ST-HSCs) or the number of MPPs. Figure 3 H). In summary, these findings suggest that CDSCs play a crucial role in restoring the irradiated BM niche, thereby promoting hematopoietic remodeling.

[0061] CDSCs regenerate BM endothelial and stromal cells in damaged BM niches. To further investigate the mechanism by which CDSCs revitalize the irradiated BM niche, a mouse model was established, which involved local irradiation of a single tibia followed by in situ injection of purified CDSCs and BM cells. Figure 4 A). After 3-6 weeks, flow cytometry analysis showed that CDSCs differentiated into CD45. - TER119 - CD31 - Triple-negative cells (i.e., total BMSCs), CD51 + BMSCs, endothelial cells, and perivascular cells (Nestin) + and Lepr + cell)( Figure 4 B). Compared with BMT, CDSCs showed enhanced differentiation into endothelial cells and BMSCs (BMT). Figure 4 C). BMSCs derived from CDSCs exhibited morphological similarities to healthy BMSCs, and they demonstrated higher proliferative capacity in vitro than irradiated BMSCs. Compared to BMT and the control group, CDSC transplantation significantly increased the number of vascular endothelial cells in the BM, even exceeding the levels observed in healthy mice. Figure 4 D). In addition, BMSCs and CD51 in CDSC receptors + BMSCs and Nestin + The total number of BMSCs was significantly higher than that of the control group. Figure 4 EG). Although for Lepr + No significant differences were observed in BMSCs. Figure 4 In summary, these findings suggest that CDSCs can differentiate into endothelial cells and BMSCs within the damaged BM niche, thereby enhancing the recovery of irradiated endothelial cells and BMSCs.

[0062] (3.5) Cultured CDSCs exhibited the BM matrix phenotype and enhanced hematopoietic recovery. To evaluate the therapeutic potential of CDSCs, CDSCs were cultured and expanded in vitro (P2, P3 passages) and sorted by flow cytometry, followed by treatment of sublethal irradiated mice with cultured CDSCs or BM cells. Results showed that, compared with controls, cultured CDSCs significantly accelerated the recovery of erythrocytes (RBCs), platelets (PLTs), and leukocytes (WBCs) in peripheral blood. Figure 5 AC).

[0063] Notably, in femoral transplantation experiments involving lethally irradiated mice, co-transplantation of cultured CDSCs with MPPs effectively rescued the mice. Conversely, mice in the groups receiving co-transplantation of MSCs with MPPs and those receiving only MPPs did not survive. Figure 5D). For co-transplantation with HSCs, the survival rate and chimerism level were comparable to those of HSCs alone. Figure 5 Mechanistically, cultured CDSCs inhibited apoptosis in Lin-BM cells one week after radiation injury. Figure 5 FG, and inhibited DNA damage in MPPs three weeks after radiation ( Figure 5 Previous studies have shown that radiation can induce senescence in HSCs, and high expression of CD150 indicates senescence in LT-HSCs. In this study, cultured CDSCs inhibited senescence in LT-HSCs and maintained a normal proportion by 4 weeks, along with increased MPP, CMP, and GMP.

[0064] To determine if there were any differences between cultured CDSCs and primary CDSCs, we performed single-cell RNA sequencing on both groups of cells. Based on UMAP analysis, the primary and cultured CDSCs were separated into two cell populations (…). Figure 5 J). Analysis of gene expression related to BMSCs, vascular endothelial cells, and chondrocytes in primary and cultured CDSCs showed that cultured CDSCs highly expressed BMSCs markers such as CXCL12, Pdgfra, Prrx1, Kitl, Spp1, Acta2, as well as endothelial cell markers such as Ly6a, Vcam1, and Emb, and osteoblast markers such as Col1a1, but the expression of chondrocyte markers such as Sox9 and Acan was decreased. Figure 5 K). Pseudo-time series analysis showed that cultured CDSCs possessed potential developmental trajectories for BM niche-related genes, while chondrocyte developmental trajectories were reduced (K). Figure 5 LM). Flow cytometry confirmed that approximately 50% of cultured CDSCs expressed markers of CXCL12-rich reticular (CAR) cells, and 60% expressed nestin. + Cell markers (Nestin) + 40% of the cells expressed the endothelial cell marker Sca1. The expression of bone marrow stromal markers increased with passage number of CDSCs. These findings indicate that cultured CDSCs exhibit BM stromal characteristics with reduced cartilage characteristics compared to primary CDSCs. Overall, cultured CDSCs not only restored the BM niche but also inhibited apoptosis and DNA damage in damaged HSCs, thereby promoting hematopoietic recovery.

[0065] Example 2: Application of CDSCs in the treatment of aplastic anemia A mouse model of aplastic anemia (AA) was established as described in a previous study (JA Huntington, A. Faille, FI Ustok, Open Biol 2025, 15(1), 240193.), using mice of similar weight. Briefly, 8-week-old CByB6F1 mice (BALB / cBy×C57BL / 6F1) were obtained from Vital River Laboratory Animal Technology Ltd. 5 × 10⁵ Gy was injected intravenously via the intraorbital vein 4–6 hours after pre-irradiation (5 Gy). 6 Lymph node (LN) cells were transplanted into mice. The lymph node cells were isolated from lymph nodes using conventional techniques. Intact lymph nodes were harvested, ground using a syringe plunger and cell filter, and the resulting cell suspension was obtained through mechanical dissociation, followed by centrifugation (primarily B and T lymphocytes). To treat aplastic anemia using cultured CDSCs, P2-P3 generation CDSCs were bilaterally injected intramedullary on day 0 (5 × 10⁻⁶ cells per cell). 5 5 × 10⁶ CDSCs were introduced into mice with aplastic anemia. For treatment using purified primary CDSCs, purified primary CDSCs (5 × 10⁶ CDSCs / mouse) were introduced on day 1. 5 (1 mouse / mouse) was injected intraorbitally into aplastic anemia mice. For aplastic anemia treated with cultured CDSCs or purified primary CDSCs in combination with cyclosporine A (CsA) or with CsA alone, CsA (HY-B0579, MCE, China) was dissolved in DMSO (ST038, Solarbio, China) and corn oil, and administered intraperitoneally to aplastic anemia mice at a dose of 1 mg / mouse (approximately 50 μg / g / day) daily from days 4 to 8. The CDSCs to CsA ratio was 5 × 10⁻⁶. 5 Quantity: 1mg × 5 = 5 × 10 5 Quantity: 5 mg. All data were collected 12–16 days post-treatment based on the survival status of mice with aplastic anemia.

[0066] Aplastic anemia is a disease characterized by the deterioration of the blood microenvironment (BM), manifested as pancytopenia and BM hypoplasia. Therefore, cultured CDSCs can be considered a potential treatment option for aplastic anemia. This study established a mouse model of aplastic anemia and treated mice with cultured CDSCs or primary CDSCs combined with CsA. Figure 6 A). Although using cultured CDSCs alone slightly prolonged the survival rate (compared to the AA model group), the improvement was not significant. Using CsA alone had a similar effect to the AA model group. Combining CDSCs with CsA significantly improved the survival rate. Figure 6B). Compared with CsA alone or untreated aplastic anemia mice, co-treatment with CsA enhanced RBC and PLT recovery (B). Figure 6 CD). Co-treatment also increased total BM cells, LSKs, and MPPs ( Figure 6 E), inhibited BM CD4 + and CD8 + T cell infiltration ( Figure 6 FG), and reduced BM cell apoptosis (BM(CD4) + and CD8 + (excluding T cells) Figure 6 J). Similar effects were observed in the spleen and peripheral blood. Figure 6 These results indicate that cultured CDSCs have the potential to treat aplastic anemia.

[0067] In summary, BM failure involves damage to both hematopoietic and non-hematopoietic cells within the BM microenvironment. CDSCs exhibit differentiation into endothelial cells and perivascular cells (including nestin). + Cells, CAR cells and Lepr + CDSCs possess the potential to differentiate into osteoblasts and other cells. These differentiated cells are capable of secreting additional pro-hematopoietic cytokines. Furthermore, CDSCs can inhibit apoptosis and reduce DNA damage in damaged HSPCs, known as rescued HSPCs (including RHSCs and RHPCs), while enhancing the hematopoietic function of donor-derived HSPCs (including DHSCs and DHSPCs). In summary, CDSCs effectively restore the function of damaged BM niches.

[0068] Example 3: Results of a study on the functional consistency of CDSCs from different sources (1) Isolation, purification and in vitro culture characteristics of human CDSCs Human CDSCs were initially obtained from costal cartilage specimens from drug-induced abortions, using the same dissociation method as in mice, thus obtaining primary human CDSCs. Specifically, the enzyme solution consisted of 0.2% type II collagenase dissolved in DMEM / F12. First, the costal cartilage was placed in an appropriate amount of enzyme solution and digested at 90 rpm for 20 minutes, followed by washing with PBS. Subsequently, a second enzymatic digestion was performed at 90 rpm for 35 minutes. Finally, the CDSCs were mechanically dissociated, resuspended in PBS containing 2% FBS, and then sorted into CD45 cells by flow cytometry. - CD51 +Cells were used to obtain purified human CDSCs. Type II collagenase specifically degrades type II collagen in collagen fibers, causing costal cartilage dissociation and the release of cells from the costal cartilage, including various cell types. The cells used in this protocol were those containing the CD45 surface marker of these dispersed and released cells. - CD51 + . cells.

[0069] This technical solution does not involve the acquisition process of costal cartilage specimens. After the costal cartilage specimens are ex vivo, human CDSCs are obtained through in vitro enzymatic digestion. The obtained human CDSCs are then further utilized for applications in the treatment of related diseases. The culture conditions for the obtained human CDSCs are the same as those for mouse CDSCs, and they can be cultured and passaged in vitro for expansion. Specifically, purified human CDSCs (CD45) are... - CD51 + Cells were seeded into minimum essential medium (HyClone, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Thermo Scientific, USA) and cultured in 6 cm cell culture dishes. Cells were cultured at 37°C in a 5% CO2 incubator, with the medium refreshed 2-3 times per week and passaged every 5 days.

[0070] Therefore, human CDSCs demonstrate high efficiency and simplicity in in vitro expansion, possessing both commercial application and large-scale production capabilities. From a cell source perspective, the core of this technical solution lies in the subsequent utilization of already acquired human CDSCs, without involving sample acquisition. Human CDSCs can be expanded in large quantities in vitro (ensuring a reliable cell source), providing a foundation for the practicality of this solution. In terms of in vitro expansion, human CDSCs exhibit excellent operability, requiring no complex special environments; the expansion method is simple and easy to implement, enabling efficient in vitro culture, passage, and expansion. This characteristic of stable in vitro expansion overcomes the limitation of "relying solely on primitive cells directly taken from the human body." Like general stem cells, they can be prepared on a large scale through standardized culture procedures, laying a solid foundation for commercial applications (such as cell therapy and drug development), highlighting their practical value.

[0071] (2) Consistency of gene expression and in vitro culture conditions of CDSCs from different sources Single-cell sequencing analysis was used to compare gene expression patterns between mouse-derived CDSCs and human-derived CDSCs at the molecular level. The results showed that mouse-derived CDSCs and human-derived CDSCs exhibited similar cell clustering. Figure 7A), and the characteristic genes (SOX9, SNORC, CNMD, MGP, MANT3, EPYC) of CDSCs from both sources were expressed identically. Figure 7 B). Simultaneously, analysis of the correlation between gene expression in human and mouse CDSCs revealed a positive correlation between the gene expression of CDSCs from the two sources. Figure 7 C). Further analysis of the cell morphology and growth of human and mouse CDSCs revealed that human and mouse CDSCs exhibited consistent cell morphology and similar proliferation rates during in vitro culture. Figure 7 (D and E). In summary, these all indicate that human CDSCs have similar gene expression characteristics and cell proliferation patterns to mouse CDSCs.

[0072] (3) Consistency of bone marrow microenvironment repair capacity among CDSCs from different sources Further research is needed to investigate the stem cell nature of human CDSCs and whether cultured human CDSCs possess the potential for bone marrow microenvironment repair, thereby applying them to the treatment of bone marrow disorders. The specific procedure for the in vitro induction of trilineage differentiation of human CDSCs is as follows: Chondrogenic differentiation: 5×10 5 CDSCs were seeded into 15 mL centrifuge tubes to generate chondrocytes. First, the cells were washed with chondrocyte induction differentiation premix (OSIER, Chondrocyte Induction Differentiation Kit, MUXMX-90041). Next, chondrocyte induction differentiation medium was gradually added, and the cells were cultured at 37°C and 5% CO2. The medium was changed every 2-3 days, allowing the cells to differentiate for 26 days. The induction process was stopped when the chondrocytes reached a diameter of 1.5-2 mm. After removing the medium, the chondrocytes were washed with PBS and fixed with 4% PFA for at least 30 minutes. Subsequently, the chondrocytes were stained with 1% Alsin Blue solution at 37°C for 1 hour, and then washed three times with PBS.

[0073] Myogenic differentiation: 20,000 CDSCs were suspended in DMEM / F12 medium and cultured at 37°C with 5% CO2. When the cells reached 80% confluence, they were seeded into 35 mm culture dishes or 6-well plates and cultured for another 2 days in myocyte differentiation medium consisting of DMEM / F12 and MvoCult™ 10-fold amplification supplement (OSIER, 05985). The medium was changed every 2–3 days throughout the myogenic differentiation process. Immunofluorescence and qRT-PCR analyses were performed to assess the expression of relevant molecules.

[0074] Osteogenic differentiation: 2×10 5CDSCs were seeded in 6-well plates coated with 0.1% gelatin (OSIER, GLT-11301). These cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (Aikeli Biotechnology, FND500) and incubated at 37°C and 5% CO2. When the cell density reached 70%, induction differentiation medium (OSIER, MUXMX-90021) was added, with replenishment every 2-3 days over 23 days. After induction differentiation, the medium was removed, and the cells were washed with PBS. The cells were then fixed with 4% cycloheximide for at least 30 minutes and stained with Alizarin Red for 10 minutes. Images of the stained cells were taken using a microscope.

[0075] Adipogenic differentiation: Adipogenesis-induced differentiation medium (Oricell, MUXMX-90031) was used. CDSCs were cultured at 2×10⁶ cells / cells. 4 Cells were seeded at a density of [number] cells / cm² in 6-well plates, and 2 mL of DMEM / F12 complete medium containing 10% fetal bovine serum was added to each well. These cells were cultured at 37°C and 5% CO₂, with the medium changed every 2-3 days. When cells reached 100% adherence, they were cultured in adipogenesis-induced differentiation medium A for 3 days, followed by adipogenesis-induced differentiation medium B for 1 day. After 15 days of induction, the medium was removed from the 6-well plates, cells were washed three times with PBS, and fixed with 4% PFA for 30 minutes at room temperature. The fixative was then removed, and the cells were washed three times with PBS. 2 mL of Oil Red O staining working solution was added to each well, and the cells were incubated at room temperature for 30 minutes. Afterward, the cells were washed 4-5 times with PBS, and then imaged using a microscope. In vitro induction experiments confirmed that human CDSCs could differentiate into bone, cartilage, fat, and fibrous tissue, indicating that human CDSCs possess trilineage differentiation capacity. Figure 8 A).

[0076] Meanwhile, human CDSCs were cultured in DMEM / F12 medium containing 10% fetal bovine serum (Aikeli Biotechnology, FND500) at 37°C and 5% CO2, using the same method as mouse CDSCs in vitro. Cells began expressing bone marrow stromal cell markers from generation P1, and the proportion of various cell types was then detected by flow cytometry. It was found that in vitro cultured human CDSCs could express multiple bone marrow stromal cell markers, and the distribution of these markers was highly consistent with that of mouse CDSCs. Figure 8 B).

[0077] More specifically, most surviving cultured human CDSCs are able to differentiate into CD45. - TER119 - CD31- Triple-negative cells (i.e., total BMSCs) showed that 47.5% of the total BMSCs expressed nestin-positive cell markers. + Cells). In the hematopoietic system, BMSCs are the core cellular components constituting the hematopoietic microenvironment. Nestin + Nestin cells, as an important subset of BMSCs, play a crucial role in maintaining HSCs, differentiating hematopoietic cells, and regulating hematopoietic homeostasis. + Cells maintain the self-renewal and survival of hematopoietic stem cells (HSCs) and regulate hematopoietic cell differentiation and lineage balance by constructing the hematopoietic microenvironment, secreting regulatory factors, and engaging in direct cell-cell interactions. They also initiate repair mechanisms during hematopoietic stress (such as injury and infection), making them key regulators of hematopoietic homeostasis and regeneration. This characteristic also makes them a potential target for research into the treatment of hematopoietic-related diseases (such as aplastic anemia and hematopoietic failure).

[0078] Of all BMSCs, 63.7% expressed the endothelial cell marker Sca1 (stem cell antigen 1). + BMSCs are an important component of the hematopoietic microenvironment, regulating the survival, self-renewal, and differentiation of HSCs through direct or indirect effects; Sca1 + BMSCs play a central role in angiogenesis and injury repair by expressing endothelial-related markers. + BMSCs, as an important subgroup of BMSCs that possesses both endothelial-related characteristics and mesenchymal stem cell functions, have core roles including: constructing hematopoietic niches to support hematopoietic homeostasis, participating in angiogenesis and repair, promoting regeneration and repair of multiple tissue injuries, and regulating immune balance.

[0079] Of the total BMSCs, approximately 55% were CXCL12-rich reticular cells (TNCs). - CD51 + Sca1 - CD140a + As a core subset of CAR cells that highly express CXCL12, these cells play a crucial role in regulating the microenvironment during hematopoietic reconstitution (such as the recovery of hematopoietic function after bone marrow transplantation and bone marrow regeneration after chemotherapy / radiotherapy). Their functions revolve around the homing, survival, self-renewal, and differentiation balance of hematopoietic stem cells (HSCs).

[0080] This demonstrates that human CDSCs can differentiate into various cell populations that reconstruct the hematopoietic microenvironment, thus playing a role in promoting hematopoietic reconstitution. Following the same method, mouse CDSCs were cultured in vitro, and the results showed ( Figure 8C): Most surviving cultured mouse CDSCs are able to differentiate into CD45. - TER119 - CD31 - Triple-negative cells (i.e., total BMSCs) comprise approximately 60% of total BMSCs, while nestin-positive cells (Nestin) are also present. + The cells are markers for endothelial cell markers, with approximately 40% expressing Sca1 and approximately 50% being CAR cells that highly express CXCL12. These three cell types are the three main types of bone marrow stromal cells, each playing a corresponding supporting role in the reconstruction of the hematopoietic microenvironment.

[0081] Through the above data comparison, it can be clearly observed that human CDSCs and mouse CDSCs share highly common characteristics when differentiating into cell populations that promote hematopoietic reconstitution. Specifically, this can be analyzed from two levels: overall expression patterns and subdivided cell types. From the overall expression patterns, the two show significant consistency. After in vitro culture, the expression patterns of cell surface markers in human CDSCs are completely consistent with those in mouse CDSCs, both stably expressing characteristic markers corresponding to three cell types: CXCL12-rich reticular cells, endothelial cells, and nestin-positive cells. This fully demonstrates that human and mouse CDSCs share the same molecular basis in their core ability to differentiate into hematopoietic remodeling-related cell populations, providing a solid common basis for cross-species research.

[0082] In terms of the proportion of different cell types, the two show a high degree of consistency in key indicators: CXCL12-rich reticular cells (TNCs) - CD51 + Sca1 - CD140a +The expression rate of this cell type in human CDSCs was 55%, which is very close to the 56.8% in mouse CDSCs. This highly similar proportion clearly shows that CDSCs from both sources have almost identical differentiation potential in key cell populations supporting the structural stability of the hematopoietic microenvironment. BMSCs expressing the endothelial cell marker (Sca1, stem cell antigen 1): The proportion of endothelial cell marker expression in human CDSCs was 63.7%, and in mouse CDSCs it was 44.7%. Although the values ​​differ, both can differentiate into a certain proportion of BMSCs expressing the endothelial cell marker, and both possess the basic ability to participate in vascular repair-related processes. This is an important commonality in their differentiation of this cell type. The higher proportion of endothelial cell marker expression in human CDSCs suggests that human CDSCs may have a superior vascular repair function. Nestin-positive cells (Nestin...) + The expression rate of this marker was 47.5% in human CDSCs and 62.7% in mouse CDSCs. Although the proportions differed, a considerable proportion of cells from both sources expressed this marker. Both CDSCs possessed the ability to maintain the self-renewal of hematopoietic stem cells (HSCs), regulate the differentiation balance of hematopoietic cells, and stabilize hematopoietic homeostasis. This is their core commonality in this function.

[0083] In summary, human and mouse CDSCs exhibit highly similar characteristics when differentiating into cell populations that promote hematopoietic remodeling. Both their overall expression patterns and the differentiation capacity of key cell types suggest that cells from both sources have similar functions and can be used to promote hematopoietic remodeling, treat bone marrow failure, treat radiation damage, treat aplastic anemia, and treat other potential hematological diseases related to BM niche dysfunction. The above studies demonstrate that human and mouse CDSCs exhibit highly consistent functional characteristics in bone marrow microenvironment repair. In terms of bone marrow microenvironment repair, both can differentiate into cell subpopulations with specific functions (such as CAR cell subpopulations highly expressing CXCL12, Nestin...). + Cells, Sca1 +Human CDSCs (cells, etc.) participate in the construction and repair of the bone marrow's reticular scaffold structure, secrete extracellular matrix components, and synergistically repair damaged vascular networks, providing stable physical support and microenvironmental signals for hematopoietic stem cells (HSCs). The consistency of these functions stems from the essentially identical differentiation potential of human and mouse CDSCs; both can differentiate into key cell subpopulations that play a crucial role in bone marrow microenvironment repair. Simultaneously, both exhibit significant stem cell characteristics. After in vitro culture, human CDSCs, like mouse CDSCs, can express large amounts of bone marrow stromal cell markers, further confirming their biological characteristics as stem cells. Therefore, human CDSCs possess a highly consistent function with mouse CDSCs in bone marrow microenvironment repair, providing important experimental evidence and theoretical support for their application in the clinical treatment of hematopoietic-related diseases.

[0084] (4) Summary of functional consistency of CDSCs from different sources By comparing human and mouse CDSCs from multiple dimensions, the study powerfully revealed the high degree of functional consistency between the two.

[0085] At the molecular level, single-cell sequencing analysis revealed significant similarities. Mouse-derived CDSCs and human-derived CDSCs showed remarkably similar cell clusters, and the expression of their characteristic genes (SOX9, SNORC, CNMD, MGP, MANT3, EPYC) was consistent. Furthermore, gene expression correlation analysis showed a positive correlation between gene expression in CDSCs from the two sources.

[0086] Cellular-level comparisons also support this conclusion. Human and mouse CDSCs exhibited consistent cell morphology and similar proliferation rates during in vitro culture. This indicates that they are virtually indistinguishable in their fundamental cell growth and development behaviors, demonstrating the similarity of their underlying cellular regulatory mechanisms.

[0087] In the investigation of stem cell nature and bone marrow microenvironment repair potential (differentiation potential study), in vitro induction of trilineage differentiation of human CDSCs confirmed that they can differentiate into bone, cartilage, fat, and fibrous cells, exhibiting trilineage differentiation capacity. Furthermore, in vitro cultured human CDSCs can express multiple bone marrow stromal cell markers. Correspondingly, in vitro culture of mouse CDSCs showed a similar proportional distribution to human CDSCs and could be directed to differentiate into key supporting stromal cells in the bone marrow niche, promoting hematopoietic reconstitution.

[0088] In summary, comparisons at the molecular, cellular morphology, and functional differentiation levels fully demonstrate that human and mouse CDSCs have essentially identical functions. Therefore, human CDSCs possess the potential to promote hematopoietic reconstitution, treat bone marrow failure, radiation damage, aplastic anemia, and other potential hematological diseases related to BM niche dysfunction, providing highly promising new ideas and solutions for the clinical treatment of these diseases.

[0089] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of costal cartilage-derived stem cells in the preparation of formulations for the treatment of bone marrow failure, characterized in that, Rib chondrogenic stem cells are cells derived from rib cartilage and having CD45 as a surface marker - CD51 + . The costal cartilage-derived stem cells were prepared by the following method: costal cartilage was digested and dissociated using type II collagenase, and then resuspended in phosphate buffer containing fetal bovine serum to obtain an enzymatically separated cell collection; then, cells that did not express CD45 but expressed CD51 on the surface were screened by flow cytometry to obtain costal cartilage-derived stem cells. Costal cartilage-derived stem cells are used for directed differentiation into supporting stromal cells in the bone marrow niche; supporting stromal cells include bone marrow mesenchymal stem cells, perivascular cells, endothelial cells, and osteoblasts; Costal cartilage-derived stem cells are used to secrete hematopoietic cytokines.

2. The application of the costal cartilage-derived stem cells according to claim 1 in the preparation of a formulation for treating bone marrow failure, characterized in that: The formulation includes costal cartilage-derived stem cells and other stem cells; the other stem cells are hematopoietic stem cells and / or pluripotent progenitor cells; Every 5×10 5 Each costal cartilage-derived stem cell corresponds to ≥50 hematopoietic stem cells; every 5 × 10 5 Each costal cartilage-derived stem cell corresponds to ≥1000 pluripotent progenitor cells.

3. The application of the costal cartilage-derived stem cells according to claim 2 in the preparation of a formulation for treating bone marrow failure, characterized in that: The bone marrow failure mentioned refers to bone marrow failure caused by radiation damage.

4. The use of the composition formed by the costal cartilage-derived stem cells and cyclosporine A according to any one of claims 1-3 in the preparation of a medicament for treating aplastic anemia.

5. The use of the composition formed by costal cartilage-derived stem cells and cyclosporine A according to claim 4 in the preparation of a medicament for treating aplastic anemia, characterized in that: The ratio of costal cartilage-derived stem cells to cyclosporine A was 5 × 10⁻⁶. 5 Quantity: 5mg.

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