Use of CD133-positive endothelial-like stem cells in preparation of medicament for promoting endothelial cell differentiation and vascular regeneration

Abstract

Use of CD133-positive endothelial-like stem cells in the preparation of a medicament for promoting endothelial cell differentiation and vascular regeneration. The medicament comprises a medicament for preventing aging, a medicament for treating progeria, or a medicament for restoring vascular vitality. Furthermore, a combination with pamidronate can improve health status and extend lifespan.

Description

Application of CD133-positive endothelial-like stem cells in the preparation of drugs that promote endothelial cell differentiation and angiogenesis Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the use of CD133-positive endothelial-like stem cells in the preparation of drugs that promote endothelial cell differentiation and angiogenesis. Background Art

[0002] Tissue degeneration is a typical feature of aging. A small number of stem cells or progenitor cells present in specific tissues are called tissue-specific stem cells (TSCs). They are able to self-renew and differentiate into tissue-specific cells, thereby maintaining tissue homeostasis. The hematopoietic system is a representative of tissues with a high renewal rate and has a high demand for tissue-specific stem cells, namely hematopoietic stem cells (HSCs); a single HSC can repopulate the entire blood system. Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) are two other prominent types of hematopoietic stem cells in the adult bone marrow (BM). Although MSCs can be easily expanded in vitro and show great potential for clinical application, the role of EPCs is still under investigation.

[0003] Asahara et al. first identified the presence of human peripheral blood endothelial progenitor cells (EPCs), which promote angiogenesis. Following endothelial injury, cytokines and growth factors, such as VEGF, SDF-1, G-CSF, and estrogen, mobilize EPCs from the basement membrane into the peripheral circulation, where they seed at the site of injury and mediate neovascularization. Typical EPC enrichment requires 1 to 4 weeks of in vitro culture. Depending on the culture duration, two main types of EPCs can be obtained: bone marrow angiogenic cells (MACs), which require 4 to 7 days of culture, and endothelial colony-forming cells (ECFCs), which require 14 to 21 days of culture. Bone marrow angiogenic cells have a weak proliferative capacity and express high levels of CD14, MAC-1, and CD11C. They also mark monocytes and macrophages but express low levels of the endothelial marker VE-cadherin (CDH5) and the stem cell markers CD133 and c-Kit15. In contrast, endothelial colony-forming cells (ECFs) are highly proliferative and positive for CD34, CD105, CD146, CD31, and VE-cadherin. Both bone marrow angiogenic cells and ECFs exist in humans, whereas only bone marrow angiogenic cells have been reported in mice. Despite being extensively studied for over two decades, in vivo evidence for EPCs remains scarce.

[0004] Accumulating evidence suggests that a decline in the function and / or number of tissue-specific stem cells contributes to the reduced regenerative capacity seen in aging and age-related diseases. In particular, the function and / or number of hematopoietic and mesenchymal stem cells decline with age. In a mouse model of Hutchinson-Gilford Progeria Syndrome (HGPS), primarily caused by mutations in LMNA, there is a marked premature depletion of mesenchymal, hematopoietic, hair follicle, and muscle stem cells. An age-related decline in the number and function of endothelial progenitor cells has also been demonstrated, which may be a major contributor to the reduced repair capacity of the vascular endothelium (VE). However, it remains unclear whether the decline, depletion, or dysfunction of tissue-specific stem cells is a cause, rather than a consequence, of aging, and whether replenishment of healthy and youthful tissue-specific stem cells can rejuvenate aging tissues / organs and promote longevity. Technical issues

[0005] One of the purposes of the embodiments of the present application is to provide the use of CD133-positive endothelial-like stem cells in the preparation of drugs that promote endothelial cell differentiation and angiogenesis, aiming to solve the problem that there is no application of CD133-positive endothelial-like stem cells in the prior art in drugs related to promoting endothelial cell differentiation and angiogenesis. Technical Solutions

[0006] To solve the above technical problems, the technical solutions adopted in the embodiments of the present application are:

[0007] In a first aspect, the invention provides the use of CD133-positive endothelial-like stem cells in the preparation of a drug for promoting endothelial cell differentiation and angiogenesis.

[0008] In some embodiments, the drug that promotes endothelial cell differentiation and angiogenesis includes at least one of a drug for preventing aging and a drug for treating premature aging.

[0009] In some embodiments, drugs that promote endothelial cell differentiation and angiogenesis include drugs that restore vascular vitality.

[0010] In some embodiments, the drug further comprises: an inhibitor of the FDPS cholesterol metabolism signaling pathway, wherein the inhibitor comprises pamidronate.

[0011] In a second aspect, an embodiment of the present application provides a drug for preparing a drug that promotes endothelial cell differentiation and angiogenesis, wherein the drug includes CD133-positive endothelial-like stem cells.

[0012] In some embodiments, the drug further comprises: an inhibitor of the FDPS cholesterol metabolism signaling pathway.

[0013] In some embodiments, the inhibitor comprises: pamidronate.

[0014] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.

[0015] In some embodiments, the carrier comprises at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

[0016] In some embodiments, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersions, and syrups. Beneficial effects

[0017] The beneficial effect of the use of CD133-positive endothelial-like stem cells provided in the first aspect of the present application in the preparation of drugs that promote endothelial cell differentiation and angiogenesis is that CD133-positive endothelial-like stem cells can improve the microvascular system, reduce weight loss and osteoporosis, and thus delay premature aging or physiological aging. Therefore, the provided CD133-positive endothelial-like stem cells can be used as raw material drugs for diseases such as promoting endothelial cell differentiation and angiogenesis, providing a theoretical basis to support the use of stem cells to treat progeria and normal aging.

[0018] The second aspect of the present application provides a beneficial effect of a drug for preparing a drug for promoting endothelial cell differentiation and angiogenesis, which includes CD133-positive endothelial-like stem cells. Since CD133-positive endothelial-like stem cells can improve the microvascular system, reduce weight loss and osteoporosis, and thus delay premature aging or physiological aging, the provided drug includes CD133-positive endothelial-like stem cells, which can achieve the effect of preventing and treating aging and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or exemplary technical descriptions. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0020] FIG1 is an analysis diagram of bone marrow mononuclear cells differentiating into endothelial cells in distant organs provided in an embodiment of the present application;

[0021] (a) Schematic diagram of the lineage tracing strategy: Recipient mice were irradiated with X-rays and transplanted with bone marrow mononuclear cells (BMNCs) from rosa26-mT / mG mice. Four weeks after bone marrow transplantation (BMT), recipient mice underwent unilateral femoral artery ligation (FAL). Three weeks after FAL, mice were sacrificed for analysis of tdTomato-positive endothelial cells (ECs).

[0022] (b) Bone marrow reconstitution rate was determined by flow cytometry of bone marrow and peripheral blood cells.

[0023] (c) Representative fluorescence images showing donor-derived CDH5-positive and tdTomato-positive (anti-red fluorescent protein, RFP) endothelial cells in cardiac tissue. Scale bar, 20 μm.

[0024] (d) Representative fluorescence images showing donor-derived CD31-positive and tdTomato-positive (anti-RFP) endothelial cells in muscle tissue. Scale bar, 20 μm.

[0025] (e) Representative fluorescence images showing donor-derived CD31- and tdTomato-positive endothelial cells in adjacent aortic tissue sections. Scale bar, 100 μm.

[0026] Five mice were analyzed. Data represent mean ± standard error.

[0027] FIG2 is an analysis diagram of irradiation-induced DNA damage and endothelial cell renewal provided in an embodiment of the present application; in the figure,

[0028] (a) Representative fluorescence images of post-irradiation mouse heart tissue co-stained with anti-γH2AX and anti-CDH5. Scale bar, 20 μm. Three mice were analyzed (non-IR represents no irradiation, 8h post-IR represents 8 hours post-irradiation, and 24h post-IR represents 24 hours post-irradiation).

[0029] (b) Representative fluorescence images showing co-staining of anti-RFP (tdTomato) and anti-CDH5 / CD31 in isolated heart (top) and muscle (bottom) tissue from bone marrow-transplanted mice using rosa26-mT / mG bone marrow mononuclear cells. Scale bar, 20 μm. Quantitative data are mean ± standard error, analyzed from three mice. (Merge represents the overlap of CDH5 and RFP fluorescence signals.)

[0030] FIG3 is a diagram showing the detection and analysis of donor-derived endothelial cells in the liver, lungs, and kidneys provided in an embodiment of the present application;

[0031] (a) Representative fluorescence images showing donor-derived CDH5-positive and RFP-positive endothelial cells in liver tissue.

[0032] (b) Representative images of lung donor-derived CD31-positive and RFP-positive endothelial cells.

[0033] (c) Representative images of kidney donor-derived CD31-positive and RFP-positive endothelial cells.

[0034] Scale bar, 20 μm. Three mice were analyzed.

[0035] DAPI represents the use of 4',6-diamidino-2-phenylindole as a fluorescent dye.

[0036] Figure 4 shows 3D visualization of CDH5 / RFP (tdTomato) co-staining in heart (a), liver (c), and kidney (d), and CD31 / RFP co-staining in muscle (b), as provided in the Examples of this application. Scale bar, 20 μm. Analysis was performed on three mice.

[0037] DAPI represents the use of 4',6-diamidino-2-phenylindole as a fluorescent dye.

[0038] FIG5 is a cell spectrum analysis diagram of mouse heart tissue after mT / mG bone marrow transplantation provided in an example of the present application.

[0039] Flow cytometric analysis of tdTomato (Tom)-positive, CD31-positive, and tdTomato (Tom) and CD31-double-positive cells in heart tissue of mice following rosa26-mT / mG bone marrow transplantation. FITC CD31-positive; early childhood development, tdTomato-positive. Analysis was performed on five mice.

[0040] FIG6 is an analysis diagram of donor-derived endothelial cells expressing GFP in heart and aorta tissues provided in an example of the present application; in the diagram:

[0041] (a) Schematic diagram of the lineage-tracing strategy: Recipient mice were irradiated with a lethal dose of X-rays and transplanted with bone marrow mononuclear cells from H11-mG / mR mice; 4 weeks after bone marrow transplantation, the recipient mice underwent unilateral femoral artery ligation. 3 weeks after unilateral femoral artery ligation, the mice were sacrificed for study. (b) Flow cytometric analysis shows the proportion of donor-derived GFP- and CD31-double-positive endothelial cells in heart tissue from mice transplanted with H11-mG / mR bone marrow mononuclear cells. GFP positivity is shown in the FITC channel; CD31 positivity is shown in the PE channel. (c) Representative fluorescence images show donor-derived CD31 (red)-GFP double-positive endothelial cells in aortic tissue sections. Five mice were analyzed.

[0042] FIG7 is a flow cytometry sorting result and microscopic analysis diagram of the differentiation of bone marrow transplant cells marked by AAV-mediated Cre expression provided in the examples of the present application;

[0043] (a) Schematic diagram of the lineage tracing strategy: Recipient mice were irradiated with X-rays and transplanted with bone marrow mononuclear cells from rosa26-mT / mG mice. Four weeks later, the recipient mice underwent unilateral femoral artery ligation. Three weeks later, AAV1-ICAM2-Cre particles were injected via the tail vein. Four weeks later, the mice were sacrificed, and tissues were collected for flow cytometric analysis. (b) Schematic diagram of the flow cytometric analysis strategy. (c) Flow cytometric analysis of fluorescent cells in heart tissue from mice transplanted with rosa26-mT / mG bone marrow treated with AAV1-ICAM2-Cre or AAV injection. The FITC channel displays the GFP signal; the ECD channel displays the tdTomato signal. (d) Flow cytometric analysis of fluorescent cells in muscle, liver, lung, and kidney tissue from mice transplanted with rosa26-mT / mG bone marrow treated with AAV1-ICAM2-Cre or AAV particles. The FITC channel displays the GFP signal; the ECD channel displays the tdTomato signal. (e) Representative fluorescence images of donor-derived GFP-positive, tdTomato-positive, and GFP- and tdTomato-double-positive cells in heart sections from mice transplanted with rosa26-mT / mG bone marrow treated with AAV-ICAM2-Cre viral particles. Five mice were analyzed.

[0044] FIG8 is a flow cytometry sorting and microscopic analysis diagram of Cre-expressing bone marrow transplant cells induced by the present invention; ...

[0045] (a) Schematic diagram of the lineage tracing strategy: Recipient mice were irradiated with X-rays and transplanted with bone marrow mononuclear cells from H11-mG / mR and Cdh5-CreERT mice. Four weeks after bone marrow transplantation, the recipient mice underwent unilateral femoral artery ligation. Three weeks after femoral artery ligation, the mice were intraperitoneally injected with tamoxifen for five days. Seven days later, the mice were euthanized, and multiple tissues were collected for flow cytometric analysis. (b) Schematic diagram of the flow cytometric analysis strategy. (c) Flow cytometric analysis of fluorescent cells in the heart, liver, kidney, and muscle tissues of H11-mG / mR and Cdh5-CreERT bone marrow-transplanted mice treated with or without tamoxifen. GFP positivity is shown in the FITC channel; tdTomato positivity is shown in the ECD channel. (d) Representative fluorescence images of donor-derived tdTomato- and GFP-double-positive cells in the heart tissue of tamoxifen-treated H11-mG / mR and Cdh5-CreERT bone marrow-transplanted mice.

[0046] FIG9 is an analysis diagram of the distribution of donor-derived cells in Tie2-Cre recipient mice provided in the examples of the present application.

[0047] (a) Schematic diagram of the lineage tracing strategy: Tie2-Cre recipient mice were irradiated with X-rays and transplanted with bone marrow mononuclear cells from rosa26-mT / mG mice. Four weeks after bone marrow transplantation, the recipient mice underwent unilateral femoral artery ligation. Three weeks after unilateral femoral artery ligation, the mice were sacrificed for study. (b) Representative fluorescence images showing co-staining of anti-RFP (tdTomato) and EGFP (top), anti-RFP and CDH5 (center), and anti-EGFP and CHD5 (bottom) in heart tissue isolated from irradiated Tie2-Cre mice transplanted with rosa26-mT / mG bone marrow mononuclear cells. Scale bar, 20 μm. Three mice were used for this study. (c) Representative fluorescence images showing co-staining of anti-RFP (tdTomato) and EGFP (top) and anti-RFP and CD31 (bottom) in muscle tissue isolated from irradiated Tie2-Cre mice transplanted with rosa26-mT / mG bone marrow mononuclear cells. Scale bar, 20 μm. Three mice were used in this study. (d) Representative images showing tdTomato and EGFP fluorescence (left) and co-staining of rosa26-mT / mG bone marrow mononuclear cells with anti-Cre and CD31 (right) in the aortic arch of Tie2-Cre mice transplanted with bone marrow. Scale bar, 100 μm. Three mice were used in this study.

[0048] FIG10 is an analysis diagram showing that donor-derived endothelial cells rarely originate from macrophages in an embodiment of the present application, in which:

[0049] (a) Representative fluorescence images showing co-staining of rosa26-mT / mG bone marrow mononuclear cells with anti-RFP (tdTomato) and F4 / 80 (top) and anti-cdh5 and F4 / 80 (bottom) in isolated cardiac tissue from mice after bone marrow transplantation. The percentage of F4 / 80-positive cells among donor-derived RFP-positive cells is shown (right). Scale bar, 20 μm.

[0050] (b) Representative fluorescence images showing co-staining of rosa26-mT / mG bone marrow mononuclear cells with anti-RFP (tdTomato) and F4 / 80 (top) and anti-CD31 and F4 / 80 (bottom) in isolated muscle of mice after bone marrow transplantation. The percentage of F4 / 80-positive cells among donor-derived RFP-positive cells is shown (right). Scale bar, 20 μm.

[0051] (c) Representative fluorescence images showing rosa26-mT / mG bone marrow mononuclear cells co-stained with anti-CD31 and F4 / 80 in the aortic arch of mice after bone marrow transplantation. F4 / 80-positive cells were detected only in the aortic endothelium. Scale bar, 100 μm.

[0052] Data are mean ± standard error. A total of 4 mice were examined.

[0053] FIG11 is an analysis diagram of a unique cell population in CD133-labeled bone marrow mononuclear cells provided in an example of the present application, in which:

[0054] (a) Cell types (clusters) of whole mouse bone marrow mononuclear cells and CDH5-positive bone marrow-derived endothelial cells.

[0055] (b) Pseudo-time trajectory analysis of SC_1, SC_2, and SC_3 subclusters.

[0056] (c) UMAP visualization of cell sources: whole mouse bone marrow mononuclear cells, CDH5-positive endothelial cells in the bone marrow cavity, and CD133-positive endothelial-like cells.

[0057] (d) Pseudo-time trajectory analysis of four stem cell subsets and endothelial cells marked by CD133.

[0058] FIG12 is an analysis diagram of CD133-positive endothelial cells and angiogenic potential provided in the examples of the present application, in which:

[0059] (a) Single-cell pseudo-time analysis of transcriptomes of whole mouse bone marrow mononuclear cells and CDH5-positive BM niche endothelial cells. Numbers represent pseudo-time proximity, not chronological order. Stem cell cluster 2 is more closely related to cluster 3, which is further divided into subclusters SC_1, SC_2, and SC_3. (b) Pseudo-time analysis shows the evolutionary trends of clusters EC, SC_1, SC_2, and SC_3, with cluster EC being more closely related to SC_2. (c) Cluster SC_2 has the most enriched gene pathways with unique expression relative to other bone marrow mononuclear cell clusters (Supplementary Figure 10, d). Enrichment of cell surface proteins. (d) Gene expression heatmap analysis of genes shown in (c) and genes used in the literature to identify endothelial progenitor cells, i.e., CD34-positive endothelial progenitor cells characterized in SC_1, 2, 3, and endothelial cells. (e) UMAP visualization of single-cell transcriptomes of CD133-positive endothelial-like stem cells, whole mouse bone marrow mononuclear cells, and CDH5-positive bone marrow-derived endothelial cells. CD133-positive endothelial-like cells clustered into CD133_1-4. Three mice were selected for analysis. (f) Pseudo-time analysis of EC subcluster EC_S and endothelial-like cell subcluster CD133_1. (g) CD133-positive endothelial-like cells labeled with red fluorescently labeled acetylated low-density lipoprotein (Dil-acLDL) and oleander agglutinin 1 (UEA-1). Three mice were examined. Scale bar, 50 μm. (h) In vitro Matrigel chamber experiments showed that CD31-positive Cdh5-positive cells had tubular structures, while such structures were not observed in CD133-negative bone marrow mononuclear cell Matrigel chambers. Scale bar, 20 μm. Arrows indicate tubular structures. (i) Representative microcirculatory images (left) and blood flow recovery quantification (right) after hindlimb ischemia in mice treated with CD133-positive endothelial-like cells, CD133- bone marrow mononuclear cells, or no endothelial-like cells / bone marrow mononuclear cells. The sample size was 4 per group. p values ​​were calculated by two-way ANOVA. (j) Representative fluorescence images showing CDH5-positive endothelial cells and tdTomato-positive (anti-red fluorescent protein, RFP) donor-derived cells in muscle tissue. Four mice were used for the experiment. Scale bar, 20 μm. (k) Blood flow recovery rate on day 14 after hindlimb ischemia in mice treated with CD133-positive, CD133-positive MPO-positive, CD133-positive MMP9-positive bone marrow mononuclear cells, or phosphate-buffered saline. The number of samples per group was 4 or 5. p values ​​were calculated by Tukey's multiple comparison test. Data represent the mean ± standard error.

[0060] FIG13 is a gene expression profile analysis diagram of CD133-positive endothelial-like cells provided in the examples of the present application.

[0061] FIG14 is an analysis diagram showing that the regeneration of CD133-positive endothelial-like cells provided in the examples of the present application can improve the aging of premature aging mice and extend their lifespan, in which:

[0062] (a) From Lmna G609G / + The percentage of CD133-positive endothelial-like cells in bone marrow mononuclear cells isolated from WT mice (n=5) and WT mice (n=5).

[0063] (b)Lmna + / + Blood flow recovery rate after unilateral femoral artery ligation in wild-type (WT) C57BL / N mice treated with CD133-positive endothelial-like stem cells and LmnaG609G / G609G CD133-positive endothelial-like stem cells. p values ​​were calculated by two-way ANOVA.

[0064] (c)Lmna + / + CD133-positive endothelial-like cells and drug-loaded Lmna f / f Blood flow recovery rate after unilateral femoral artery ligation in Tie2-Cre treated mice. p values ​​were calculated by two-way ANOVA.

[0065] (d) Representative fluorescence images showing CDH5-positive endothelial cells and tdTomato (RFP)-positive donor-derived cells in heart tissue, and CD31-positive endothelial cells and RFP-positive donor-derived cells in muscle tissue. Three mice were used for this experiment.

[0066] (ef) Representative 3D reconstruction images of trabecular bone determined by Micro-CT (e) and quantitative analysis (f) of bone volume / tissue volume (BV / TV), trabecular number, and trabecular spacing (SP) in WT endothelial-like cell- or drug-treated LmnaG609G / G609G mice.

[0067] (g) Endothelial-like stem cells treated with and without LmnaG609G / G609G and Lmna + / + Mouse body weight.

[0068] (h) Endothelial-like stem cells treated with and without LmnaG609G / G609G and Lmna + / + Lifespan of mice.

[0069] Scale bar, 20 μm. Data represent mean ± standard error. Survival analysis was performed using the log-rank test (Mantel-Cox test).

[0070] FIG15 is an analysis diagram showing that the regeneration of CD133-positive endothelial-like stem cells can improve the aging of physiologically aged mice and prolong their lifespan, as provided in the examples of the present application. In the diagram:

[0071] (a) Percentage of CD133-positive endothelial-like stem cells in bone marrow mononuclear cells of young (3-month-old, Y) and old (27-month-old, O) mice (n=5). p values ​​were calculated using Student's Student t-test.

[0072] (b) Blood flow recovery after unilateral femoral artery ligation in wild-type (WT) C57BL / N mice treated with CD133-positive endothelial-like stem cells at young (Y, 3 months) and old (O, 27 months). p values ​​were calculated by two-way ANOVA.

[0073] (c) Representative fluorescence images show CDH5-positive endothelial cells (heart) and CD31-positive endothelial cells (muscle) from young (4 months old, sample number 3) and old (26 months old, sample number 3) mice treated or untreated (sample number 3) with endothelial-like cells isolated from young (3 months old) mice. Capillary density (number of capillaries per square millimeter) is shown.

[0074] (d) Representative images of CD31-positive endothelial cells and tdTomato (RFP)-positive donor-derived cells in cardiac tissue. Three mice were used for this experiment.

[0075] (e) Representative images of CD31-positive endothelial cells and tdTomato (RFP)-positive donor-derived cells in muscle tissue. Three mice were used for this experiment.

[0076] (f) Micro-CT data of isolated femurs from mice of different ages, treated with CD133-positive endothelial-like cells from young mice or vehicle.

[0077] (g) Endurance of aged mice (18 months) treated with CD133-labeled endothelial-like cells isolated from young mice or vehicle.

[0078] (h) Body weight of female mice treated or untreated with CD133-positive endothelial-like stem cells. Open squares indicate the period of body weight change between the two groups.

[0079] (i) Lifespan of CD133-positive endothelial-like stem cells and subclusters treated and untreated mice.

[0080] Scale bar, 20 μm. Data represent mean ± standard error. Survival analysis was performed using the log-rank test (Mantel-Cox test).

[0081] FIG16 is a three-dimensional visualization analysis diagram of endothelial cells stained with co-immunofluorescence provided in an embodiment of the present application, in which:

[0082] (a, b) Three-dimensional visualization of co-staining of CDH5 and RFP (tdTomato) in the heart (a) and CD31 and RFP in the muscle (b). Scale bar, 20 μm. Three mice were used in this experiment.

[0083] FIG17 is an analysis diagram of the damage of endothelial-like cell function caused by upregulation of FDPS provided in the examples of the present application, in which;

[0084] (a) UMAP visualization of the single-cell transcriptomes of CD133-positive endothelial-like cells enriched in young (3 months old, sample number 3) and aged (18.5 months old, sample number 3) mice. The left panel shows cell affiliation; the right panel shows cell identification. (b) Violin plots show transcriptome levels of Prom1, Fdps, Tspo, and Anxa1 genes in the CD133_1 cluster from young and aged mice. (c) Quantitative PCR analysis of Prom1, Fdps, Tspo, and Anxa1 transcript levels in CD133-labeled endothelial-like stem cells and CD133 bone marrow mononuclear cells from young (3 samples) and aged (3 samples). P21, Ceacam1, Ki67, and Ccnd1 levels were used as controls. (d) The isoprenoid biosynthesis pathway centered on Fdps. Pamidronate is a bisphosphonate that inhibits FDPS activity. (e-f) Overexpression of Fdps in young CD133-labeled endothelial stem cells via rAAVDJ impairs their angiogenic capacity, as determined by unilateral femoral artery ligation (f, 3 mice / group; g, 4-6 mice / group). p values ​​were calculated by two-way ANOVA. (g) Blood flow recovery after hindlimb ischemia in mice treated with young or old endothelial cells, with or without pamidronate (PAM). p values ​​were calculated by two-way ANOVA. Data represent mean ± standard error.

[0085] FIG18 is a diagram showing differential gene expression analysis between young and old CD133-positive endothelial-like stem cells provided in the examples of the present application.

[0086] FIG19 is an analysis diagram showing that pamidronate improves aging and prolongs lifespan in physiologically aging mice as provided in the examples of the present application, wherein;

[0087] (a) Percentage of CD133-positive endothelial-like cells in bone marrow mononuclear cells isolated from pamidronate (PAM)-treated aged mice and control mice (27 months of age, n=4 of 5). (b) Quantitative PCR analysis of p16 and Il-6 transcript levels in CD133-positive endothelial-like stem cells from pamidronate-treated aged mice and control mice (24 months of age, n=4–6). (c) Blood flow recovery in a unilateral femoral artery ligation model treated with CD133-positive endothelial-like stem cells isolated from PAM-treated aged mice or control mice (26 months of age). P values ​​were calculated using two-way ANOVA. (d) Representative fluorescence images showing CDH5-positive endothelial cells in heart tissue from aged mice (26 months of age, n=4) treated or untreated. Capillary density (capillaries per square millimeter) is shown. (e) Running activity of aged mice treated with pamidronate or control vehicle. (fh) Lifespan of mice treated or not treated with pamidronate. (f) Female mice; (g) Male mice; (h) Mixed sexes. Data are mean ± standard error. Survival analysis was performed using the log-rank test (Mantel-Cox test). Modes for Carrying Out the Invention

[0088] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit this application.

[0089] Prior to this, the applicant has demonstrated that endothelial cell dysfunction can lead to systemic aging in mice. In the course of the study, it was found that CD133 marks potential endothelial progenitor cells in the bone marrow, that is, endothelial-like cells also have similar effects. Therefore, there is an urgent need to provide the relevant effects of CD133-positive endothelial-like stem cells.

[0090] In a first aspect, the embodiments of the present application provide the use of CD133-positive endothelial-like stem cells in the preparation of a drug for promoting endothelial cell differentiation and angiogenesis.

[0091] The beneficial effect of the use of CD133-positive endothelial-like stem cells provided in the first aspect of the embodiment of the present application in the preparation of a drug that promotes endothelial cell differentiation and angiogenesis is that CD133-positive endothelial-like stem cells can improve the microvascular system, reduce weight loss and osteoporosis, and thus delay premature aging or physiological aging. Therefore, the provided CD133-positive endothelial-like stem cells can be used as raw material drugs for diseases such as promoting endothelial cell differentiation and angiogenesis, providing a theoretical basis to support the use of stem cells to treat progeria and normal aging.

[0092] In some embodiments, the drug that promotes endothelial cell differentiation and angiogenesis includes at least one of a drug for preventing aging and a drug for treating premature aging.

[0093] In some embodiments, drugs that promote endothelial cell differentiation and angiogenesis include drugs that restore vascular vitality.

[0094] In some embodiments, the drug further comprises: an inhibitor of the FDPS cholesterol metabolism signaling pathway, wherein the inhibitor comprises pamidronate.

[0095] A second aspect of the embodiments of the present application provides a drug for preparing a drug that promotes endothelial cell differentiation and angiogenesis, wherein the drug includes CD133-positive endothelial-like stem cells.

[0096] The beneficial effect of a drug for preparing a drug for promoting endothelial cell differentiation and angiogenesis provided in the second aspect of an embodiment of the present application is that: the drug for preparing a drug for promoting endothelial cell differentiation and angiogenesis provided includes positive endothelial-like stem cells. Since CD133-positive endothelial-like stem cells can improve the microvascular system, reduce weight loss and osteoporosis, and thus achieve the purpose of delaying premature aging or physiological aging, the provided drug includes CD133-positive endothelial-like stem cells, which can achieve the effect of preventing and treating aging and other related diseases.

[0097] In some embodiments, the drug further comprises: an inhibitor of the FDPS cholesterol metabolism signaling pathway.

[0098] In some embodiments, the inhibitor comprises: pamidronate.

[0099] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.

[0100] In some embodiments, the carrier comprises at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

[0101] In some embodiments, the addition of a diluent is mainly used to increase the weight and volume of the drug to facilitate molding and dosing. In the embodiments of the present application, the diluent is selected from but not limited to at least one of starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose.

[0102] In some embodiments, the addition of a wetting agent can moisten the material to generate sufficient viscosity to facilitate granulation. In this embodiment, the wetting agent is selected from, but not limited to, at least one of water, ethanol, and glycerin. In some embodiments, the addition of a binder can aggregate and bind non-viscous or less-viscous materials into granules. In this embodiment, the binder is selected from, but not limited to, at least one of hydropropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), starch slurry, and syrup.

[0103] In some embodiments, coating agents and colorants are added to improve the appearance of tablets, increase the stability of the drug, mask the unpleasant odor of the drug, and change the appearance of the particles. In the embodiments of the present application, the coating agent is selected from but not limited to at least one of acrylic resin, hypromellose, povidone, and cellulose acetate; the colorant is selected from but not limited to at least one of titanium dioxide, sunset yellow, and methylene blue.

[0104] In some embodiments, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersions, and syrups.

[0105] In some embodiments, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersions, and syrups.

[0106] The present application will be further described by the following experimental steps and examples, which are for illustrative purposes only and do not limit the scope of the present application.

[0107] Test operation

[0108] (1) Animals

[0109] LmnaG609G / G609G and Lmna f / f TC Tie2-Cre mice were obtained from previous experiments in our laboratory. Rosa26-mT / mG mice were provided by Dr. Jian Chen from Soochow University, China. C57BL / 6 mice, H11-mG / mR mice, and Cdh5-CreERT mice were purchased from Jiangsu, China. All mice were housed and handled according to protocols approved by the Shenzhen University Committee on the Use of Animals in Teaching and Research.

[0110] (2) Hindlimb ischemia

[0111] Hindlimb ischemia was achieved by unilateral femoral artery ligation (FAL). Briefly, mice were anesthetized with 2% isoflurane inhaled via an isoflurane delivery system (EZVET, China). A 1-cm skin incision was made on the left hindlimb, and the neurovascular pedicle was visualized under light microscopy. A ligature was placed in the left femoral artery proximal to the branches of the superficial epigastric artery and anterior to the saphenous artery. The accessory branches between the femoral artery and the ligature were then excised. The skin was closed with 4-0 sutures, and erythromycin ointment was applied postoperatively to prevent wound infection. Preoperative and postoperative blood flow recovery was assessed using a dynamic microcirculatory imaging system (Teksqray, Shenzhen, China). Relative blood flow recovery was expressed as the ratio of ischemic to nonischemic blood flow. At least three mice were included in each experimental group.

[0112] (III) RNA isolation and quantitative PCR analysis

[0113] Cells were lysed using Trizol RNAiso Plus (Takara, Japan), and total mRNA was extracted according to the manufacturer's instructions. cDNA was reverse transcribed using 5× Evo M-MLV RT Master Mix (Accurate Biology, China). Gene expression levels were measured using Hieff qPCR SYBR Green Master Mix (Yeasen, China) on a CFX Connect real-time PCR detection system (Bio-Rad, USA). All primer sequences are shown in Table 1.

[0114] Table 1

[0115] (IV) Immunofluorescence staining

[0116] Mouse tissue cryosections (10 μm thickness) were fixed in 4% PFA for 15 minutes, permeabilized with 0.3% Triton X-100 for 15 minutes, blocked with 5% bovine serum albumin for 30 minutes, and incubated with appropriately diluted primary antibodies overnight at 4°C. After washing three times with phosphate-buffered saline to remove the primary antibody, the tissue sections were incubated with Alexa Fluor-conjugated secondary antibodies for 1 hour at room temperature. After washing three times with phosphate-buffered saline, the sections were mounted with antifade mounting medium containing DAPI and observed under a Zeiss LSM880 confocal microscope. All antibodies are listed in Table 2 below.

[0117] Table 2

[0118] (5) Ultrasound

[0119] Mice in each group underwent transthoracic echocardiography (IU22, Philips) after inhalation of isoflurane. Echocardiographic analysis obtained heart rate, cardiac output, left ventricular posterior wall dimension (LVPWD), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular ejection fraction (LVEF), and left ventricular shortness fraction. Each experimental group included at least three mice.

[0120] (6) Bone density measurement

[0121] Mice were kept at 4°C overnight, and data were collected using micro-CT scanning (Scanco Medical, micro-CT100). Micro-CT parameters evaluated included trabecular number (1 / mm), trabecular spacing (SP) (mm), and bone volume / tissue volume (BV / TV%). Each group included at least three mice.

[0122] (7) Durability operation test

[0123] Fatigue resistance was monitored using a rotarod treadmill (YLS-4C, Jinan Yiyan Research Institute, China). Mice were placed on a rotating track, and the rotation speed was gradually increased to 40 rpm. When the mice reached exhaustion and fell from the track, the endurance time was recorded. Each experimental group included at least three mice.

[0124] (8) Bone marrow transplantation

[0125] Bone marrow mononuclear cells were collected from 3-month-old rosa26-mT / mG mice or H11-mG / mR mice and injected into 3-month-old C57BL / 6 recipient mice via the tail vein. The lethal dose of X-ray irradiation was 9Gy. The steps include: after the mice were euthanized, the femur and tibia were separated, cut at both ends, and placed in a 0.5ml microcentrifuge tube with a hole drilled at the bottom. A 1.5ml microcentrifuge tube was placed inside a 0.5ml microcentrifuge tube, and the tube was centrifuged at 3000×g for 15 seconds. Red blood cells were removed by 450×g gradient density centrifugation (Histopaque-1083, Sigma) for 30 minutes. The bone marrow mononuclear cells were resuspended in pre-cooled phosphate buffered saline supplemented with 1% fetal bovine serum (FBS), and 1.5×10 7 Bone marrow mononuclear cells were injected into irradiated recipient mice via the tail vein. After four weeks of recovery, the recipient mice underwent unilateral femoral artery ligation (FAL). Three weeks after FAL, the mice were sacrificed for further study.

[0126] (IX) Magnetic-activated cell sorting (MACS) and cell therapy

[0127] Bone marrow mononuclear cells were obtained as described above. After centrifugation at 450×g for 5 min at 4°C, the bone marrow mononuclear cells were suspended in 80 μl MACS buffer and incubated with 20 μl anti-CD133 antibody (Miltenyi Biotec, Germany) for 15 min. They were then incubated with 20 μl MACS anti-biotin microbeads (Miltenyi Biotec) in 80 μl MACS buffer. CD133-positive endothelial-like stem cells were then obtained by magnetic selection. For endothelial-like cell therapy, 1×10 cells were collected from rosa26-mT / mG mice every other week. 6 CD133-positive endothelial-like stem cells were injected into old mice through the tail vein.

[0128] (10) Fluorescence-activated cell sorting

[0129] Bone marrow mononuclear cells were obtained as described above. After centrifugation at 450 g for 5 min at 4°C, the cells were suspended in 600 μl of MACS buffer and incubated with 5 μl of APC-CD133 antibody (Biolegend, USA) at 4°C for 30 min. CD133-positive endothelial progenitor cells were sorted using a flow cytometer (BD Biosciences, USA).

[0130] (11) Phosphate therapy

[0131] Pamidronate (PAM) (Selleckchem, China) dissolved in phosphate-buffered saline was administered to female mice at a weekly infusion of 3 mg / kg starting around 90 weeks of age. The mice's health and lifespan were closely monitored. Cardiac function and treadmill activity were measured after 8 weeks of treatment.

[0132] (XII) In vitro Matrigel test

[0133] The angiogenic capacity of CD133-positive endothelial cells was determined by in vitro matrigel assay. Briefly, 500 μL of growth factor-reduced matrigel (Corning, USA) was mixed with 5 × 10 5 CD133-positive endothelial-like stem cells or CD133 were subcutaneously injected into the mid-dorsal region of 12-week-old male mice. Two weeks later, the Matrigel inserts were surgically removed, and cryosections were prepared and subjected to immunofluorescence analysis.

[0134] (XIII) Single-cell library preparation, sequencing and mapping

[0135] CD133-positive endothelial-like stem cells (approximately 8,000–10,000 cells) were enriched by flow cytometry for single-cell sequencing. RNA was quantified by Qubit and RT-PCR on a LightCycler 96 system (Roche Life Science, Germany). Single-cell RNA was barcoded, reverse transcribed, fragmented, and amplified to generate cDNA using the Chromium Single Cell 3' Reagent Kit v2 (10× Genomics, USA). The size of the pre-amplified cDNA was determined using the Agilent High Sensitivity D5000 and D1000 Screen-Tape systems (Agilent, USA). Sequence libraries were constructed according to the manufacturer's instructions, and single-cell libraries were sequenced in a single-index custom paired-end format on a HiSeq 1500 Sequencing System (Illumina, USA). Data were compared using STAR. Based on the read map of the reference genome, unique molecular identifiers (UMIs) and the expression of each gene in each cell were calculated using Cell Ranger 2.1.0 software. The average number of reads per cell ranged from 74,323 to 92,229, and the median number of genes per cell ranged from 1,947 to 2,342.

[0136] (14) Data QC and cell type identification

[0137] Each data matrix was plotted using the “Seurat” package (v3.0.1), and data processing and visualization were performed using custom scripts in R. Genes detected in fewer than three cells and cells expressing fewer than 200 genes were removed. “Seurat-Find Variable Features” was applied to identify highly variable genes. After removing batch effects, the recombinant dataset was filtered according to the following criteria to exclude poor-quality cells: UMI count <5% or >95% or mitochondrial gene proportion >20%. Principal component analysis was performed using the “FindNeighbors” and “FindClusters” functions to perform clustering. Dimensionality was reduced using t-distributed random neighbor embedding to visualize the clustering results. Cell types were identified using marker genes of cell clusters, with the cell type results identified by the “SingleR” package as a reference.

[0138] (15) Single-cell data analysis

[0139] Unsupervised pseudo-time analysis of single cells was performed using Monocle software, and the results were used as a reference for the differentiation process of myeloid cells. Differentially expressed genes for each cell type were calculated using the "Seurat-FindMarkers" function and the "MAST" software package (v1.8.2), with an FDR-corrected p-value of 0.05. Protein-protein interaction network functional enrichment analysis was based on the "STRING" database (https: / / string-db.org / ) and visualized using "Cytoscape" (V3.6.1). Gene set enrichment analysis was based on the enrichment of KEGG and GO functions using the "ClusterProfiler" and "GSEA" packages.

[0140] (16) Statistical analysis

[0141] The statistical significance between two groups of data was determined using the t-test. One-way analysis of variance was used to test the statistical significance between multiple groups of data. Two-way analysis of variance was used to test the statistical significance between multiple time series data. Survival rates were analyzed using the log-rank test (Mantel-Cox test). All data are presented as mean ± standard deviation or mean ± standard error. A p-value of < 0.05 was considered statistically significant.

[0142] Example 1: Bone marrow mononuclear cells differentiate into endothelial cells in distant organs

[0143] Given that endothelial cells and hematopoietic cells share a common origin, it has been hypothesized that angioblasts, like endothelial progenitor cells, may originate in the bone marrow. To test this hypothesis, a lineage tracing strategy was employed (Figure 1a): bone marrow mononuclear cells (BMNCs) from rosa26-mT / mG mice expressing tdTomato and EGFP were transplanted into C57BL / 6 recipient mice irradiated with a lethal dose of X-rays (9 Gy). X-ray irradiation induced extensive DNA damage, as demonstrated by γ-H2AX fluorescence staining (Figure 2a).

[0144] The recipient mice were allowed to recover for four weeks before undergoing unilateral femoral artery ligation (FAL) to stimulate angiogenesis. Three weeks later, the mice were sacrificed for further investigation. Bone marrow regeneration was as high as 80%, and peripheral blood regeneration was close to 50% (Figure 1b).

[0145] Immunofluorescence microscopy with double staining using anti-tdTomato and either anti-CDH5 or anti-CD31 was used to examine whether donor-derived bone marrow mononuclear cells could differentiate into endothelial cells. As shown in Figure 1c and d, a large number of tdTomato-positive cells were also positive for CDH5 or CD31 in the heart and muscle tissue of bone marrow-transplanted (bone marrow-transplanted) mice (Figure 1c, d). Irradiation enhanced the turnover of endothelial cells in both heart and muscle tissue (Figure 2b). Approximately 43.7±2.2% of CDH5-positive cells in the heart were tdTomato-positive, indicating that they were derived from donor bone marrow mononuclear cells. Similarly, 57.9±3.8% of CD31-positive cells in muscle tissue were derived from donor bone marrow mononuclear cells. Parallel imaging of tdTomato-positive and anti-CD31-stained fluorescence signals in adjacent aortic tissue sections revealed that the majority of CD31-positive endothelial cells were derived from rosa26-mT / mG bone marrow mononuclear cells (Figure 1e). In contrast, donor-derived cells were barely detectable in the liver, lungs, and kidneys (Figure 3). To exclude the possibility that the spatially co-localized fluorescence signals were artifacts of image processing in which two adjacent cells were positive for tdTomato or CD31, respectively, multi-layer image acquisition and three-dimensional construction were performed. Co-localized tdTomato (shown by anti-RFP staining) and anti-CD31 / CDH5 signals were observed at the single cell level in both heart and muscle tissues, but such co-localization was hardly observed in liver and kidney tissues (Figure 4). Flow cytometry was also used to detect donor-derived endothelial cells in the heart tissue of mice after bone marrow transplantation at single-cell resolution. Approximately 30% of endothelial cells were labeled with tdTomato (Figure 5), indicating their bone marrow mononuclear cell origin.

[0146] To confirm that bone marrow-derived endothelial progenitor cells can differentiate into endothelial cells in distant organs, an independent lineage tracing mouse model was used: H11-mG / mR mice, which contain a CAG-LoxP-ZsGFP-Stop-LoxP-tdTomato cassette. Bone marrow mononuclear cells from H11-mG / mR mice were transplanted into lethally X-ray-irradiated C57BL / 6 recipient mice. The recipient mice were allowed to recover for 4 weeks before undergoing unilateral femoral artery ligation. Three weeks later, the mice were sacrificed for study (Figure 6a). Approximately 22% of endothelial cells in cardiac tissue were found to be GFP-labeled (Figure 6b). Anti-CD31 immunofluorescence staining of thoracic aorta sections revealed that a large number of endothelial cells differentiated from the zsGFP-labeled bone marrow mononuclear cells (Figure 6c).

[0147] Next, a recombinant adeno-associated virus serotype 1 (rAAV1) containing a synthetic ICAM2 promoter was synthesized to drive endothelial-specific expression of Cre. rAAV1-ICAM2-Cre particles were injected into rosa26-mT / mG bone marrow transplanted mice via the tail vein at a dose of 1×10 11 vg / 200L. Four weeks later, the mice were euthanized and tissues were collected for flow cytometry analysis (Figure 7a). If bone marrow mononuclear cells can differentiate into endothelial cells after bone marrow transplantation, tdTomato-labeled, GFP-labeled (under the long-term action of Cre recombinase), and tdTomato- and GFP-labeled cells can be observed. The results showed that in cardiac tissue, about 21% of the fluorescently labeled cells only expressed tdTomato, marking that the donor bone marrow mononuclear cells had not differentiated into endothelial cells, about 6% of the cells were positive for both tdTomato and GFP, indicating that the donor cells were differentiating, and more than 70% of the cells were only labeled with GFP, indicating that endothelial cells were differentiated from bone marrow mononuclear cells after bone marrow transplantation (Figure 7c). In contrast, the GFP signal was almost undetectable in other tissues (Figure 7d). GFP-positive cells were also detected in cardiac tissue using immunofluorescence microscopy (Figure 7e).

[0148] Furthermore, H11-mG / mR mice were crossed with the inducible Cdh5-creERT mouse line to generate mG / mR;Cdh5-Cre mice, in which Cre expression is driven by the promoter of the endothelial-specific Cdh5 gene in the presence of tamoxifen. Bone marrow mononuclear cells were collected from mG / mR;Cdh5-Cre mice and transplanted into C57BL / 6N mice irradiated with a lethal dose of X-rays. After 4 weeks of recovery, the recipient mice underwent unilateral femoral artery ligation. 3 weeks later, the mice were intraperitoneally injected with tamoxifen (70 mg / kg / day) for 5 days after bone marrow transplantation. After 7 days, the mice were euthanized and tissues were collected for flow cytometry analysis (Figure 8a). If zsGFP-labeled bone marrow mononuclear cells can differentiate into endothelial cells, they will express tdTomato under the action of tamoxifen-induced Cre, regardless of whether they express GFP. Of all fluorescently labeled cells in heart tissue, approximately 56% carried only tdTomato, representing endothelial cells differentiated from bone marrow mononuclear cells after bone marrow transplantation, and 15% carried both GFP and tdTomato, likely differentiating or just differentiating into endothelial cells (Figure 8b). In contrast, tdTomato-positive cells were barely detected in liver, kidney, and muscle tissues (Figure 8c). Frozen sections were also prepared, and cells expressing tdTomato were detected using immunofluorescence microscopy (Figure 8d).

[0149] In addition to direct differentiation, the observed results could also result from fusion of local recipient endothelial cells with donor cells, passive diffusion of fluorescent proteins to neighboring cells, and transdifferentiation of macrophages into endothelial cells. To rule out this possibility, we first used the Tie2-Cre mouse line as recipients and performed bone marrow reconstitution with rosa26-mT / mG bone marrow mononuclear cells (Figure 9a). If fusion occurs, Cre recombinase from the recipient endothelial cells will work with the rosa26-mT / mG loci in the donor bone marrow cells and activate EGFP expression. If tdTomato passively spreads, it will be detected in Cre-positive endothelial cells. tdTomato-positive endothelial cells are common in heart, muscle, and aorta tissues, while EGFP-positive endothelial cells are rare (Figure 9bd). tdTomato-positive cells lack Cre staining (Figure 9d). Finally, we examined whether the observed tdTomato-positive endothelial cells were macrophages by F4 / 80 staining. Although endothelial cells double-positive for F4 / 80 and tdTomato were observed, their frequency was very low (Figure 10). All these data suggest that potential endothelial cells exist among the basal cells in the post-bone marrow transplantation model and can differentiate into endothelial cells of distant organs to maintain endothelial homeostasis.

[0150] Example 2: CD133 marks potential endothelial progenitor cells in mouse bone marrow

[0151] Having demonstrated the EC differentiation potential of bone marrow mononuclear cells (BMMCs), we sought to further enrich for endothelial progenitor cell (EPC) subpopulations. We reasoned that EPCs should be more closely related to endothelial cells in the bone marrow. To test this hypothesis, we first used single-cell RNA sequencing data from two studies describing whole-mouse BMMCs and CDH5-positive cells in the BM cavities, designated endothelial cells. After re-clustering based on marker gene expression, the two datasets were integrated after removing batch effects. Pseudotemporal analysis of all cells revealed that BMMC clusters SC_1, SC_2, and SC_3, predicted to possess stem / progenitor cell properties, were closely related to endothelial cells (Figure 12a and Figure 11a). Further pseudotemporal analysis revealed that SC_2 possessed more progenitor cell characteristics and was predicted to differentiate into SC_1 or SC_3 (Figure 11b), suggesting that SC_2 likely represents an endothelial progenitor cell upstream of endothelial cells (Figure 12b).

[0152] Next, we examined the transcriptomes of the three clusters and other populations in the bone marrow to identify a putative molecular signature for cluster SC_2. A set of cell membrane-associated components marked the SC_2 cluster, with CD133, encoded by the Prom1 gene, being the most prominent (Figure 12c). Heat map analysis (Figure 12d) revealed genes that were consistent with those used in the literature to identify endothelial progenitor cells, indicating a CD34-positive endothelial progenitor cell signature in SC_1, 2, 3, and endothelial cells. This putative marker set was termed the CD133-positive cell signature, and its expression was compared with reported endothelial progenitor cell markers: CD45, CD14, and CD31 for myeloid angiogenic cells; CD105, CD14, and CD31 for endothelial colony-forming cells; VCAM1, ICAM1, and KDR for endothelial cells; and CD34, collectively referred to as the CD34-positive endothelial progenitor cell signature. Along the pseudo-timeline (Figure 12b), CD133-positive cell signatures were significantly expressed in SC_2, sharply decreased in SC_1, and almost disappeared in endothelial cells. Among CD34-positive endothelial progenitor cell signatures, CD14 expression was highest in SC_2, followed by SC_1, and lowest in endothelial cells. CD34 and ICAM1 were expressed to some extent in SC_1 but were almost absent in SC_2. This suggests that CD133 may mark early endothelial progenitor cells, namely CD133-positive endothelial-like cells (ECs).

[0153] Therefore, flow cytometry (FACS) was used to enrich CD133-positive bone marrow mononuclear cells from three C57BL / 6N mice. To experimentally verify the characteristics of CD133-positive endothelial-like cells, 10× Genomics single-cell RNA sequencing was performed. After quality filtering, 10,869 cells were recovered. Using the k-means clustering algorithm, CD133 endothelial-like cells were further clustered into four subpopulations: CD133_1, CD133_2, CD133_3, and CD133_4 (Figure 12e). Although Prom1 was ubiquitously expressed in all four subpopulations, the Mpo gene encoding myeloperoxidase was primarily expressed in the CD133_1 cluster, and Mmp9 was primarily expressed in the CD133_4 cluster (Figure 12e and Figure 13). On the pseudo-time axis, the CD133_1 cluster was more strongly associated with endothelial cells (Figure 12f and Figure 11c).

[0154] The functional properties of CD133-positive endothelial-like stem cells were further studied. It is known that endothelial progenitor cells absorb low-density lipoprotein (LDL) and urea oleracea agglutinin 1 (UEA-1). More than 95% of fresh flow cytometry-sorted CD133-positive endothelial-like cells were positive for bisphenol-conjugated acetylated low-density lipoprotein and urea oleracea agglutinin 1 (Figure 12g), indicating that they have endothelial differentiation potential. In order to study the potential of CD133-positive endothelial-like cells in angiogenesis, an in vitro matrigel chamber experiment was performed. The positive endothelial-like stem cell matrigel chamber showed tubular structures containing CD31-positive Cdh5-positive cells, which were not observed in the CD133-bone marrow mononuclear cell matrigel chamber experiment (Figure 12h). Then, CD133-positive endothelial-like cells and bone marrow mononuclear cells from rosa26-mT / mG mice were enriched using magnetic activated cell sorting (MACS) and injected into mice given unilateral femoral artery ligation via the tail vein. Dynamic microcirculation imaging system analysis showed that after supplementation with CD133-positive endothelial-like stem cells, the blood flow recovery rate on day 14 was significantly increased from 50% to nearly 85%, while it was only 65% ​​after treatment with CD133-negative bone marrow mononuclear cells (Figure 12i). Donor-derived tdTomato-expressing endothelial cells were frequently observed at the site of injury, with approximately 34.6±1.4% of CD31-positive cells being tdTomato-positive (Figure 12j). Mice treated with CD133-positive endothelial-like cells and CD133 and MPO double endothelial-like cells, representing the CD133_1 cluster, showed comparable blood flow recovery rates on day 14 after unilateral femoral artery ligation (Figure 12k). Treatment with endothelial-like cells that are double-positive for CD133 and MMP9, representing CD133_4, only restored blood flow. Overall, these data indicate that CD133-positive endothelial-like stem cells in the bone marrow (mainly represented by the CD133 and MPO double-positive subpopulation) promote the formation of new blood vessels.

[0155] Example 3: CD133-positive endothelial-like cells improve premature aging characteristics in mice

[0156] Endothelial dysfunction accelerates aging and shortens life span in a mouse model of progeria syndrome: Lmna f / f； The lifespan of TC was prolonged. We hypothesized that endothelial dysfunction is caused by defects in endothelial progenitor cells and that supplementation of endothelial progenitor cells might extend the lifespan of the model mice. To test these hypotheses, we first analyzed the expression of Lmna by flow cytometry. G609G / + The number of CD133-positive endothelial-like cells in Lmna and WT control mice was significantly higher than that in control mice. G609G / +The number of CD133-positive endothelial-like cells in mice increased significantly (Figure 14a). Next, CD133-positive endothelial-like cells were sorted, and their function in angiogenesis was studied in a unilateral femoral artery ligation model. Compared with the control group, the neovascularization ability of CD133-positive endothelial-like stem cells from the systemic progeria syndrome model-LmnaG609G / G609G mice was impaired by about 30% (Figure 14b), indicating that the function of LmnaG609G / G609G CD133-positive endothelial-like stem cells decreased. Then, it was explored whether supplementing CD133-positive endothelial-like stem cells can rescue Lmna f / f； Indeed, orthotopic injection of CD133-positive endothelial-like stem cells isolated from rosa26-mT / mG mice completely restored the expression of Lmna f / f； Neovascularization after injury in TC mice ( FIG. 14 c ).

[0157] LmnaG609G / G609G mice begin to show signs of aging at 2 months of age and die 4-6 months after birth. Next, we questioned whether young and healthy CD133-positive endothelial-like stem cells can rejuvenate aging blood vessels and improve premature aging in progeria mice. To test these hypotheses, CD133-positive endothelial-like stem cells from 3-month-old and healthy rosa26-mT / mG mice were enriched by magnetic-activated cell sorting (MACS). Then, starting from 3 months of age (before the earliest death event), LmnaG609G / G609G mice were injected with 1×10 6 CD133-positive endothelial-like stem cells were expressed for one month. Fluorescence microscopy detected donor-derived tdTomato-positive endothelial cells in the heart and muscle, but not in the aorta (Figure 14d). After supplementation with endothelial progenitor cells, LmnaG609G / G609G mice showed a significant increase in trabecular number and a significant decrease in trabecular spacing (Figure 14e,f). EPC treatment also alleviated premature weight loss (Figure 14g). The average lifespan of progeria mice was extended by nearly 20% (from 21 weeks to 25 weeks). Taken together, these data indicate that CD133-positive endothelial-like stem cells promote angiogenesis and extend the lifespan of the progeria mouse model.

[0158] Example 4: CD133-positive endothelial-like stem cells rejuvenate aged blood vessels and promote lifespan

[0159] Vascular aging / dysfunction is a hallmark of aging; however, its causal relationship still lacks experimental support. Compared with young mice (3 months old, Y), the number of CD133-positive endothelial-like stem cells in old mice (27 months old, O) increased (Figure 15a), while the angiogenesis ability of endothelial-like cells (O) was significantly impaired compared with endothelial-like cells (Y) (Figure 15b). Compared with young mice, the number of + endothelial cells in the heart and muscle of old mice was significantly reduced (Figure 15c). Then, it was studied whether supplementing CD133-positive endothelial-like stem cells could enhance the ability of angiogenesis. To this end, 1×10 cells enriched in 3-month-old rosa26-mT / mG mice were added every other week. 6 Endothelial-like cells were injected into 18-month-old mice via the tail vein. After 8 months of endothelial cell supplementation, the capillary density (CD31-positive endothelial cells) in the gastrocnemius muscle increased significantly from 234.5±9.9 / mm 2 Increased to 393.3±14.8 / mm 2 (p<0.05), the latter and young mice (3 months old, 409.9±13.1 / mm 2 Correspondingly, the capillary density of the heart (CDH5-positive endothelial cells) also increased significantly after endothelial-like cell treatment, from 2051.5±42.6 / mm 2 Increased to 2532.9±157.4 / mm 2 (p<0.05), compared with young mice (3 months old, 2570.3±101.6 / mm 2 tdTomato-positive cells were continuously observed in heart and muscle tissues 8 months after the first injection of endothelial-like cells ( FIG. 15 d, e and FIG. 16 ).

[0160] Next, the overall aging characteristics of aged mice treated with endothelial cells were analyzed. The ratio of bone volume (BV) to tissue volume (TV) and the number of trabeculae gradually decreased, while trabecular separation / spacing increased with age. Endothelial cell treatment alleviated the exacerbation of bone loss, even reaching significance in BV / TV%. Endothelial cell treatment also increased running endurance (Figure 15fh). In addition, body weight did not change until late aging, when untreated mice began to die. After supplementation with CD133-positive endothelial stem cells, the median lifespan was extended by 10%, from 115 weeks to 126 weeks. In addition, the lifespan-extending effects of CD133+MPO+ and CD133-positive MPO-negative mmp9-negative endothelial stem cell subsets were tested. Endothelial stem cells with both CD133 and MPO positive also extended their lifespan to a similar extent as CD133-positive endothelial stem cells. In contrast, the supplementation of the CD133+ MPO+ MMP9+ subpopulation only slightly increased lifespan, but did not reach significance (Fig. 15i), indicating that CD133-positive endothelial-like stem cells, especially the CD133 and MPO+ subpopulation, can rejuvenate aging blood vessels and promote the lifespan of mice.

[0161] Example 5: FDPS underlies aging of endothelial-like cells

[0162] In order to understand the potential mechanisms driving endothelial cell aging and functional decline, single-cell transcriptomes of CD133-positive endothelial-like cells screened from bone marrow by flow cytometry were obtained and compared from young (3-month-old) and old (18.5-month-old) mice. 8,362 cells and 10,597 cells were collected from young and old mice, respectively (Figure 17a). Among them, about 454 genes were significantly upregulated and 90 genes were significantly downregulated in the endothelial-like cells sorted from old mice (Figure 18). Further functional enrichment analysis showed that there were two significantly enriched pathways in old CD133-positive endothelial-like stem cells: ribosome biogenesis and oxidative phosphorylation. The 200 genes with the highest expression upregulation in these two pathways were selected for protein-protein interaction network analysis, and the gene expression of the four subpopulations of CD133_1, CD133_2, Cd133_3 and CD133_4 in young and old mice was compared. Three genes, Fdps, Anxa1, and Tspo, were significantly and consistently upregulated in all four groups of aged endothelial stem cells (Figure 17b, c), as verified by quantitative RT-PCR: Fdps, Anxa1, and Tspo were all highly expressed in CD133-positive bone marrow stem cells (Figure 17d). Although the expression levels of Anxa1 and Tspo were significantly enhanced in aged CD133-positive endothelial stem cells and CD133-negative bone marrow mononuclear cells, the upregulated expression of Fdps in aged mice appeared to be more specific to CD133-positive endothelial stem cells. Therefore, further mechanistic studies focused on Fdps. CD133-positive endothelial stem cells are more proliferative than CD133-negative cells because the Ki67 level in CD133-positive endothelial stem cells is much higher than that in CD133-negative cells, while the Ccnd1 level in CD133-positive endothelial stem cells is much higher than that in CD133-negative cells.

[0163] Fdps encodes farnesyl diphosphate synthase (FDPS), which is a key enzyme in the biosynthesis of isoprenoids and catalyzes the formation of farnesyl diphosphate (FPP). Farnesyl diphosphate is a precursor of sterols, alcohols, carotenoids and ubiquinone, and is also a substrate for protein acylation and geranylation (Figure 17e). The bisphosphonate derivative pamidronate (PAM) inhibits farnesyl diphosphate synthase activity and is clinically used to treat osteoporosis. In order to determine whether increased Fdps expression leads to decreased endothelial cell function in aged mice, a recombinant AAV serotype DJ (rAAV DJ) vector was constructed, which contains an Fdps expression cassette driven by the ubiquitous CMV promoter. Fdps overexpression was observed in CD133-positive endothelial stem cells (Figure 17f). In the unilateral femoral artery ligation model, Fdps overexpression significantly impaired the new blood vessel formation ability of endothelial cells from young mice (Figure 17g). In contrast, incubation with pamidronate (PAM) significantly enhanced the neovascularization capacity of endothelial cells from aged mice, while co-treatment of pamidronate with young endothelial stem cells showed minimal additive effects (Figure 17h). Therefore, increased Fdps impairs the function of endothelial stem cells during aging.

[0164] Example 6 Pamidronate treatment extends the lifespan of mice

[0165] Next, the in vivo effects of pamidronate treatment were studied. To this end, 22-month-old female mice were injected with pamidronate (1 mg / kg body weight) or phosphate-buffered saline (PBS). After two months of treatment, the number of CD133-positive endothelial-like stem cells in pamidronate-treated mice was found to be significantly increased compared with that in control mice (Figure 19a). The aging marker genes P16 and Il-6 were significantly downregulated in the CD133-positive endothelial-like stem cells of pamidronate-treated mice (Figure 19b). Whether pamidronate treatment can enhance the function of CD133-positive endothelial-like stem cells was also studied. To this end, 18-month-old female mice were injected with pamidronate (1 mg / kg) weekly for 2 months. CD133-positive endothelial-like stem cells were then sorted from the treated and control mice by flow cytometry, and 1×10 6 In fact, the blood flow recovery rate was significantly improved in mice injected with endothelial cells isolated from pamidronate-treated mice compared with that in control mice ( Figure 19 c ).

[0166] Next, we explored whether long-term pamidronate treatment to inhibit farnesoid diphosphate synthase could rejuvenate aging tissues and promote the lifespan of mice. Elderly female mice around 90 weeks of age (~21 months) were divided into two groups and injected intraperitoneally with pamidronate (1 mg / kg) or a control solvent (phosphate buffered saline). The health status and lifespan of each group were closely monitored. After 6 months of PAM treatment, the capillary density of myocardial tissue (CDH5-positive endothelial cells) increased from 1610.24±131.32 / mm 2 Significantly increased to 1889.3±135.6 / mm 2 (p < 0.05) (Figure 19d). In addition, during the transition from darkness to light, mice treated with pamidronate showed a faster transition from activity to rest than untreated mice (Figure 19e). For mice with a survival time exceeding the median (~115 weeks; ~122 weeks), the lifespan of female mice was significantly extended from 118.5±4.3 weeks to 128.4±3.52 weeks (Figure 19f, P = 0.0371). Similarly, the therapeutic effect of pamidronate on male mice was studied. Both the median and maximum lifespans of male mice treated with pamidronate were significantly increased (Figure 19g, P = 0.02). If the two sexes are combined, lifespan is also significantly extended (P = 0.0304). Therefore, it is concluded that pamidronate treatment can prolong the lifespan of mice.

[0167] The stem cell theory of aging states that a decline in stem cell number and / or function leads to tissue regeneration defects and consequential aging. Replicative stress caused by somatic cell loss leads to a rapid depletion of adult stem cells, leading to progeria. Hematopoietic and mesenchymal stem cells are two stem cell populations found in the bone marrow with enormous clinical potential. Previously, it has been demonstrated that the number and function of mesenchymal and hematopoietic stem cells decline in mouse models of progeria. However, transplantation of mesenchymal or hematopoietic stem cells from healthy donors into these models has little beneficial effect. CD133-positive endothelial-like stem cells, potential endothelial progenitor cells, are found in the bone marrow. Consistent with the decrease in hematopoietic and mesenchymal stem cells, the number and function of CD133-positive endothelial-like stem cells decline in progeria mice. During physiological aging, although the number of CD133-positive endothelial-like stem cells increases, their function in angiogenesis is significantly impaired in mice. Rejuvenation of CD133-positive endothelial-like stem cells improves microvasculature, attenuates weight loss and osteoporosis, and extends lifespan in both progeria- and physiologically aged mice. This study provides the first evidence supporting the use of stem cells to treat progeria and normal aging.

[0168] The nomenclature and origin of endothelial progenitor cells (EPCs) are controversial. Leveraging the power of single-cell transcriptomics, studies have been able to define various populations within bone marrow mononuclear cells (BMMCs) and endothelial cells (ECs). Using pseudo-temporal analysis, CD133-positive BMMCs were found to be closely associated with endothelial cells in the bone marrow. Using the genetically engineered rosa26-mT / mG mouse model, it was demonstrated that CD133-positive EPs possess endothelial differentiation and angiogenesis capabilities. In pseudo-temporal analysis, CD133-positive EPs (cluster SC_2) moderately expressed CD14 and VCAM1 but expressed little CD34, CD105, CD146, CD31, and VE-cadherin, marking endothelial cells and endothelial colony-forming cells. Endothelial cells lost CD133 expression, while cluster SC_1 expressed moderate levels of CD14, CD34, and ICAM1. Previously, Peichev et al. found that co-expression of CD133 and VEGFR2 identified circulating endothelial progenitor cells. VEGFR2 was found to be highly expressed in CD133-positive endothelial-like stem cells. In contrast to myeloid angiogenic cells and endothelial colony-forming cells, CD133-positive endothelial-like stem cells failed to expand in vitro. Based on the collected evidence, it is speculated that CD133 may mark an early stage in the evolution of endothelial progenitor cells. Single-cell transcriptomics revealed that CD133-positive endothelial-like stem cells can be divided into four groups: CD133_1 is most closely associated with bone marrow endothelial cells and is marked by high levels of MPO, while CD133_4 is extensively labeled with MMP9. The CD133_1 cluster, which is dually positive for both CD133 and MPO, defines the primary function of CD133-positive endothelial-like stem cells in angiogenesis and lifespan extension. On the other hand, the CD133_4 cluster, consisting of CD133MMP9-double-positive bone marrow mononuclear cells, is hypothesized to be more closely related to bone marrow angiogenic cells, which in turn induce paracrine effects during angiogenesis. In contrast, CD133_2 and CD133_3, i.e., CD133+ MPO-neg MMP9-neg bone marrow mononuclear cell clusters, showed the poorest angiogenesis and lifespan extension abilities. Further in-depth investigation is needed to investigate the differences and precise roles of CD133-positive subpopulations in angiogenesis and senescence.

[0169] Further studies have shown that bone marrow-derived CD133-positive endothelial-like stem cells have angiogenic capacity in the heart, muscle, and blood vessels, but not in the liver and kidney. Resident endothelial progenitor cells may also exist in specific tissues / organs, where they mediate vascular endothelial homeostasis. In the context of aging, the replenishment of endothelial cells significantly increases capillary density in the heart and muscle.

[0170] Although the number of CD133-positive endothelial stem cells increases with age, their function declines. Single-cell analyses revealed that a cholesterol metabolism pathway centered on Fdps is crucial for endothelial cell function. Farnesyl diphosphate synthase regulates the biosynthesis of isoprenoids, phenols, ubiquinone, and cholesterol, thereby mediating protein farnesylation and / or geranylation. Here, we demonstrate that inhibition of farnesyl diphosphate synthase by pamidronate enhances the regenerative capacity of CD133-positive endothelial cells. Manipulation of Fdps gene expression using an rAAVDJ vector accelerates aging and dysfunction of endothelial stem cells. In vivo administration of pamidronate significantly delays endothelial cell senescence, enhances angiogenesis, promotes health, and extends lifespan. Prelamin A, the pathogenic factor in progeria syndrome, is farnesylated, and farnesyl transferase inhibitors (FTIs) can ameliorate progeria by inhibiting prelamin A farnesylation and aberrant localization. A recent study demonstrated that inhibition of farnesyl diphosphate synthase by pamidronate rescues premature aging. Upstream, hydroxymethylglutaryl-CoA reductase catalyzes the production of mevalonate and is therefore essential for the production of geranyl pyrophosphate. Statins can inhibit hydroxymethylglutaryl-CoA production, thereby shifting endothelial progenitor cells from the BM to the peripheral circulation and enhancing endothelial repair. This highlights the important role of the Fdps-mediated sterol metabolism pathway in regulating aging and longevity. The specific mechanisms regulating FDPS expression during aging and how the decline in farnesoid diphosphate synthase affects the function of CD133-positive cells warrant further investigation.

[0171] Overall, the results demonstrate that CD133 identifies endothelial cells with angiogenic potential in the bone marrow. Healthy and young CD133-positive endothelial-like stem cells can rejuvenate senescent blood vessels, ameliorate aging features, and promote lifespan in both progeria and physiologically aged mice. At the molecular level, the findings highlight the critical role of the cholesterol metabolism pathway, centered around the Fdps pathway, in regulating endothelial-like cell senescence / dysfunction and lifespan. Screening for chemicals targeting the FDPS pathway may benefit health and longevity. These findings also suggest the potential of stem cell-based therapeutic strategies for treating progeria and anti-aging applications.

[0172] The above are merely optional embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included within the scope of the claims of the present application.

Claims

1. Use of CD133-positive endothelial-like stem cells in the preparation of drugs for promoting endothelial cell differentiation and angiogenesis.

2. The use according to claim 1, characterized in that: The drug for promoting endothelial cell differentiation and angiogenesis includes at least one of a drug for preventing aging and a drug for treating premature aging.

3. The use according to claim 1, characterized in that The drug for promoting endothelial cell differentiation and angiogenesis includes a drug for restoring vascular vitality.

4. The use according to claim 1, characterized in that: The drug also includes: an inhibitor of the FDPS cholesterol metabolism signal pathway, wherein the inhibitor includes pamidronate.

5. A drug for preparing a drug for promoting endothelial cell differentiation and angiogenesis, characterized in that: The drug includes CD133-positive endothelial-like stem cells.

6. The drug according to claim 5, characterized in that The medicine also includes: an inhibitor of the FDPS cholesterol metabolism signal pathway, wherein the inhibitor includes: pamidronate.

7. The drug according to claim 5 or 6, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

8. The drug according to claim 7, characterized in that The carrier includes at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

9. The drug according to claim 5 or 6, characterized in that The dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, and syrups.

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