Method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells

Abstract

The invention relates to the technical field of adipose-derived mesenchymal stem cells, in particular to a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells. The method comprises the following steps: selecting proper animal mice; carrying out primary ADSCs separation on the mouse; the method comprises the following steps: performing adherent culture on stromal vascular components, and separating Sca-1 + ADSC through a magnetic bead sorting method to form ADSC-AM cells; the positive rate in the ADSC-AM is detected through flow cytometry; and detecting the proliferative activity, osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation capabilities in the ADSC-AM cells. According to the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the invention, it is innovatively found that a cell population enriched by using Sca-1 as a marker of ADSC (adipose-derived mesenchymal stem cells) has better adipose-derived stem cell characteristics, and the cell population has the advantages of being capable of efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, high in specificity, high in specificity, high in specificity, high in specificity and the like, and capable of effectively enriching Sca-1 positive adipose-derived mesenchymal stem cells. Specific surface molecular markers such as adherent growth, three-line differentiation capability and high purity; meanwhile, an ADSC-AM method is innovatively used, and the adipose-derived mesenchymal stem cells obtained by the ADSC-AM method are optimal in performance.

Description

Technical Field

[0001] The present invention relates to the technical field of adipose-derived mesenchymal stem cells, and in particular to a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells. Background Art

[0002] Adipose-Derived Stem Cells (ADSC) were first isolated from the stromal vascular fraction (SVF) of adipose tissue aspirated from the human body in 2001. SVF refers to the adipose tissue that has been processed to remove impurities such as mature adipose tissue, connective tissue, and blood, including adipose-derived stem cells.

[0003] (CD29+CD90+CD31-CD45-), endothelial progenitor cells

[0004] (CD34+CD31+CD146+CD45-), pericytes

[0005] A heterogeneous cell population consisting of components such as adipocytes (CD146+CD44+CD29+CD34-CD45-), preadipocytes (CD34+CD45-CD31-CD146-), endothelial cells (CD31+CD34-), smooth muscle cells (SMA+), lymphocytes (CD4+CD8+), and macrophages (CD45+CD14+CD34+CD206+).

[0006] Due to the lack of specific markers, the exact origin of adipose-derived mesenchymal stem cells in the stromal vascular fraction (SVF) is still controversial. In recent years, more and more evidence has revealed the close relationship between adipose-derived mesenchymal stem cells and adipose tissue blood vessels. On the one hand, studies have found that there is a subpopulation of adipose-derived mesenchymal stem cells expressing CD201high around the blood vessels of human subcutaneous fat, which regulates DACT2 through the classical Wnt signaling pathway to affect the phenotype of perivascular stem cells, confirming that the vascular adventitia is the true habitat of adipose-derived mesenchymal stem cells. Other studies have shown that adipose-derived mesenchymal stem cells are components of the vascular wall, and are located at the junction of endothelium and adipocytes in blood vessels, playing a dual role in generating adipocytes and supporting vascular supply.

[0007] Studies have shown that perivascular cells are likely to be adipose-derived mesenchymal stem cells. They share multiple characteristics, similar gene profiles, similar antigenic markers, etc. Secondly, perivascular adipose-derived mesenchymal stem cells can also differentiate into vascular endothelial cells and vascular smooth muscle cells. Using single-cell sequencing technology, it was found that the CELEC11A+PV-subpopulation of adipose-derived mesenchymal stem cells located around blood vessels is an important source of vascular smooth muscle differentiation. Adipose-derived mesenchymal stem cells are located around blood vessels and act as a storage cell population that differentiates into perivascular cells. This also explains why transplantation of stromal vascular fraction SVF can promote vascular regeneration in ischemic tissues and improve blood supply, but transplantation of mature adipocytes has poor efficacy.

[0008] Stem cell antigen Sca-1, also known as Ly-6A / E, is an 18kda glycosylphosphatidyl alcohol-anchored cell surface protein (GPI-AP) in the Ly-6 gene family. It was initially considered to be a specific marker for mouse hematopoietic cells. It was later found that in addition to the hematopoietic system, Sca-1 is also widely expressed in skeletal muscle, myocardium, liver, peripheral blood and other tissues and organs. However, it has not yet been determined whether it can be used as the main surface molecular marker for adipose mesenchymal stem cells. According to the minimum standards for mesenchymal stem cells (MSCs) published by the International Society for Cell Therapy (ISCT) in 2006: ① The cells can be cultured adherently; ② They have the potential to differentiate in three directions: adipogenic, chondrogenic and osteogenic differentiation; ③ They have some specific molecular markers, such as MSCs expressing CD44 and CD29, but not CD45, CD31 and HLA-DR. However, these markers can be co-expressed in a variety of cells, and the surface markers of adipose mesenchymal stem cells are still under development.

[0009] Mesenchymal stem cells have important immunomodulatory and tissue repair capabilities. Adipose-derived mesenchymal stem cells are an important candidate source for mesenchymal stem cell therapy and can be isolated from the stromal vascular fraction SVF. However, SVF represents a heterogeneous cell population that includes adipose-derived mesenchymal stem cells, endothelial cells, smooth muscle cells, resident monocytes and other cells. The traditional direct adhesion method can only remove some impurities, which affects the purity of the cells on the one hand, and on the other hand, the obtained adipose-derived mesenchymal stem cells have obvious heterogeneity and many unknown subpopulations, making it difficult to accurately grasp the functional characteristics of the cells. At present, the identification of markers for adipose-derived mesenchymal stem cells is still under continuous exploration and development.

[0010] To this end, a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells is designed to provide another technical solution to the above technical problems. Summary of the invention

[0011] Based on this, it is necessary to provide a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells in order to solve the technical problems raised in the above-mentioned background technology.

[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0013] A method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, comprising the following steps:

[0014] Choose the appropriate animal mouse;

[0015] Isolation of primary ADSCs from mice;

[0016] The stromal vascular fraction was cultured adherently, and Sca-1+ADSCs were separated by magnetic bead sorting to form ADSC-AM cells;

[0017] The positive rate in ADSC-AM was detected by flow cytometry;

[0018] The proliferation activity, osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation abilities of ADSC-AM cells were detected.

[0019] As a preferred embodiment of the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the present invention, the steps of isolating primary ADSCs from mice are as follows:

[0020] Euthanasia was performed by carbon dioxide asphyxiation and immersion in 70% ethanol for 3 min;

[0021] Inguinal fat was obtained and transferred to a 60 mm culture dish;

[0022] After washing with PBS, the tissue was minced into 1-2 mm^3 fragments and digested in a solution containing 0.25% type II collagenase and 0.1% type I collagenase at 37°C for 1 h;

[0023] The obtained SVF cell population was incubated with magnetic beads and antibodies, and then Sca-1 positive selection was performed in a sorter in a magnetic field.

[0024] As a preferred embodiment of the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the present invention, the steps of performing the adherent culture of ADSCs and separating Sca-1+ADSCs by magnetic bead sorting are as follows:

[0025] SVF cells were directly cultured on the wall;

[0026] SVFs were adherently cultured to the third generation, and +ADSCs were isolated by magnetic bead sorting. After purification, the culture medium was replaced every 2 days and cells were passaged at 80% confluence.

[0027] As a preferred embodiment of the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the present invention, the positive rate of ADSC-AM is detected by flow cytometry, and the steps are as follows:

[0028] ADSC-AM cells were dark stained with fluorescent antibodies for 45 min;

[0029] After dark staining, the cells were washed with a buffer containing 0.5% BSA and detected by flow cytometry.

[0030] As a preferred embodiment of the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the present invention, the steps of detecting the proliferation activity, osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation ability in ADSC-AM cells are as follows:

[0031] The proliferation activity of ADSC-AM cells was detected by cell counting reagent;

[0032] The osteogenic differentiation ability of ADSC-AM cells was detected by osteogenic differentiation reagent;

[0033] Adipogenic differentiation induction reagent was used to detect osteogenic and adipogenic differentiation in ADSC-AM cells;

[0034] The chondrogenic differentiation ability of ADSC-AM cells was detected by chondrogenic differentiation reagent.

[0035] As a preferred embodiment of the method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells provided by the present invention, the proliferation activity of ADSC-AM cells is detected by a cell counting kit, and the steps are as follows:

[0036] The third generation ADSC-AM cells were seeded in the well plate and cultured for 14 days;

[0037] The cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for 20 min, and CFU-Fs were recorded and counted.

[0038] An application of efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, applying the method of efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells described in claims 1 to 6 to human adipose tissue.

[0039] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0040] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0041] The present invention provides a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, innovatively discovering that the cell population enriched using Sca-1 as a marker of adipose-derived mesenchymal stem cells has better adipose-derived mesenchymal stem cell characteristics, such as adherent growth, tri-lineage differentiation ability (adipogenesis, osteogenic and chondrogenic) and high-purity specific surface molecular markers; at the same time, the ADSC-AM method is innovatively used, and through comparative studies, the adipose-derived mesenchymal stem cells obtained by the ADSC-AM method have the best performance; these inventions can provide an important basis for optimizing the research and application of adipose-derived mesenchymal stem cells in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 This is a schematic flow chart of the separation, purification and enrichment of ADSC-AM of adipose mesenchymal stem cells of the present invention;

[0044] Figure 2 This is a schematic diagram of the actual process of separation of adipose-derived mesenchymal stem cells according to the present invention;

[0045] Figure 3 This is a schematic diagram of the actual process of separation of adipose-derived mesenchymal stem cells according to the present invention;

[0046] Figure 4 Schematic diagram of cell proliferation ability detection obtained by three adipose mesenchymal stem cell isolation methods of the present invention;

[0047] Figure 5 A schematic diagram comparing the expression rates of ADSCs surface antigen markers obtained by the three methods of the present invention;

[0048] Figure 6 A schematic diagram of the three-lineage differentiation potential of adipose-derived mesenchymal stem cells obtained by comparing three methods of the present invention;

[0049] Figure 7 The present invention obtains human Sca-1 positive adipose-derived mesenchymal stem cells by ADSC-AM method. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 the present invention.

[0051] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0052] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0053] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0054] Embodiment 1

[0055] Reference Figure 1-Figure 6 , a method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells.

[0056] 1. Materials and Methods

[0057] 1. Animals

[0058] Specific pathogen-free (SPF) male C57BL / 6J mice aged 4 to 6 weeks were housed in temperature- and humidity-controlled individually ventilated cages (IVCs) with a 12-h light / dark cycle and received humane care. All procedures were performed according to the 3R principle to reduce animal suffering.

[0059] 2. Isolation of Primary ADSCs

[0060] C57BL / 6J mice (4-6 weeks old) were euthanized by carbon dioxide asphyxiation and immersion in 70% ethanol for 3 minutes. Inguinal fat was obtained and transferred to a 60 mm culture dish (Labselect, Hefei, China). After rinsing with PBS (Gibco, Grand Island, NY, USA), the tissue was minced into 1-2 mm^3 fragments and digested in a solution containing 0.25% type II collagenase (Gibco, Grand Island, NY, USA) and 0.1% type I collagenase (Gibco, Grand Island, NY, USA) at 37°C for 1 hour. The digestion was terminated with separation medium and the mixture was filtered through a 40 μm cell strainer. After centrifugation at 400 g for 10 min, the stromal vascular fraction (SVF) was resuspended in α-MEM medium containing 10% FBS (Gibco, Grand Island, NY, USA), 100 U / mL penicillin, 100 μg / mL streptomycin, and 250 ng / mL amphotericin B (Gibco, Grand Island, NY, USA) for further use.

[0061] 3. Adherent culture of ADSCs and MACS

[0062] 3.1 ADSC-A

[0063] In this method, SVF cells were directly cultured by adhering to the wall. After incubation for 48 hours in a 37°C 5% CO2 incubator, non-adherent cells were removed and the culture medium was replaced every 2 days. 0.25% trypsin-EDTA solution (Pricella, Wuhan, China) was used for subculturing until 80% confluence.

[0064] 3.2ADSC-M

[0065] Sca-1+ cells were separated by magnetic bead sorting (MACS) and cultured adherently. Briefly, Sca-1 biotin antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was bound to Sca-1 expressed on SVF cells and then coupled to anti-biotin microspheres (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) by incubation at 2-8°C for 15 minutes. Sca-1+SVF cells were adsorbed on MS columns (BioLegend, San Diego, CA, USA) under a strong magnetic field. When the magnetic field was withdrawn, the MS strains could be washed with MACS Buffer or PBS to obtain Sca-1+SVF cells, which were then further purified and amplified in a 37°C 5% COz incubator.

[0066] 3.3ADSC-AM

[0067] In this method, SVF was cultured to the third generation, and then MACS was performed to separate Sca-1+ADSCs. The culture medium of the purified cells was changed every 2 days, and the cells were passaged at 80% confluence.

[0068] 4. Cell Proliferation Analysis

[0069] 4.1 Cell Counting Kit-8 (CCK-8)

[0070] The third generation cells were seeded in 96-well plates (Labselect, Hefei, China) at 2×103 / well. On days 1, 2, 3, and 4, 100 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well and incubated for 1 h, and the absorbance was measured at 450 nm.

[0071] 4.2 Colony-forming unit fibroblasts (CFU-F)

[0072] The third generation cells were seeded at 2×103 / well in 6-well plates (Labselect, Hefei, China) and cultured for 14 days. The cells were then fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 30 min, stained with crystal violet (Beyotime, Shanghai, China) for 20 min, and CFU-Fs were recorded and counted.

[0073] 5. Flow Cytometry Analysis

[0074] The cells at passage 4 were stained with CD44-APC (BioLegend, San Diego, CA, USA), CD29-PE (BioLegend, San Diego, CA, USA), CD45-PE (BioLegend, San Diego, CA, USA) and CD31-PE (BioLegend, San Diego, CA, USA) antibodies for identification. The proportion of Sca-1+ cells among the three groups was compared by targeting Sca-1-PE (BioLegend, San Diego, CA, USA). In brief, the cells of the three groups were darkly stained with five fluorescent antibodies for 45 minutes, then washed with a buffer containing 0.5% BSA (BioLegend, San Diego, CA, USA), and detected by flow cytometry (CytoFLEX).

[0075] 6. Three-lineage differentiation induction

[0076] 6.1 Osteogenic differentiation

[0077] According to the instructions of the osteogenic differentiation kit (Cyagen, Guangzhou, China), the third generation aADSCs, mADSCs and amADSCs were seeded at a density of 2 × 104 cells / cm2 in a 6-well plate coated with 0.1% gelatin. Osteogenic differentiation medium was added to each well instead of normal medium, and fresh medium was replaced every 3 days when 70% confluence was reached. After 3 weeks, the cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S (pH 5.1-5.3) for 10 min. The results of osteogenic differentiation were observed and recorded under a microscope.

[0078] 6.2 Adipogenic differentiation

[0079] According to the instructions of the adipogenic differentiation kit (Cyagen, Guangzhou, China), the third generation aADSCs, mADSCs, and amADSCs were seeded at a density of 2×104 / cm2 in a 0.1% gelatin-coated six-well plate. Adipogenic differentiation medium A was added to each well, and the cells were incubated for 3 days after reaching 100% confluence, and then replaced with adipogenic differentiation medium B for 1 day. This cycle continued until sufficient lipid droplets appeared, usually within 1 to 2 weeks. The cells were then fixed with 4% paraformaldehyde and stained with pH 2.1 Oil Red O solution for 30 minutes. The results of adipogenic differentiation were observed and recorded under a microscope.

[0080] 6.3 Chondrogenic Differentiation

[0081] According to the instructions of the chondrogenic differentiation kit (Cyagen, Guangzhou, China), the 3rd generation aADSCs, mADSCs and amADSCs were transferred to a 15 mL test tube and centrifuged at 250 g for 4 minutes. The cell spheres were resuspended with chondrogenic premix and centrifuged at 150 g for 5 minutes. This procedure was repeated twice. Then the chondrogenic differentiation medium was added to the tube and replaced every 2 days. After 3 weeks of induction, the chondrogenic spheres were sliced ​​and stained. The chondrogenic differentiation results were observed and recorded under a microscope.

[0082] 7. Statistics

[0083] All experiments were repeated at least 3 to 5 times. Quantitative data were analyzed using GraphPad Prism 9.0 software (GraphPadsoftware Inc., San Diego, CA, USA). Two-way analysis of variance or one-way analysis of variance was used for comparison among the three groups, and P < 0.05 was considered statistically significant.

[0084] 2. Results

[0085] 1.amADSCs have uniform and typical MSC morphology

[0086] To intuitively describe the three methods for isolating and purifying ADSCs, the acquisition process was compiled into a diagram ( Figure 1 ) to illustrate the order, convergence and differences of different methods. All three methods start with obtaining SVF. Compared with ADSC-A, ADSC-M and ADSC-AM both added magnetic bead sorting, but the order was different: ADSC-M was subjected to magnetic bead sorting before adherent culture, and ADSC-AM was subjected to magnetic bead sorting after adherent culture. ADSC-A needs to be cultured continuously for at least 3 generations to ensure purity. Similarly, ADSC-M requires Sca-1 positive screening and then adherent purification until the third generation. After the ADSC-AM was adhered to the wall, the third generation cells were taken for Sca-1 positive selection. Therefore, there was no significant difference in the culture time of the three methods.

[0087] ADSC-A, the separated SVF was directly cultured and purified. ADSC-MA, the separated SVF was first sorted by Sca-1+ magnetic beads, and then purified by adherence. ADSC-AM, the separated SVF was first cultured and digested to obtain the third generation cells, and then sorted by Sca-1+ magnetic beads.

[0088] refer to Figure 2 , Figure 2 (A) The mouse was killed by cervical dislocation and placed in a 100mm sterile culture dish. (B) The mouse skin was dissected and fixed to expose the inguinal fat pad. (C) The inguinal fat pad was collected and transferred to another 60mm sterile culture dish and covered with isolation medium containing antibiotics. (D) The culture dish was transferred to the clean bench and washed at least three times, and impurities such as lymph nodes, fascia, and blood were carefully removed. Then, the adipose tissue was minced using sterile microscissors in a small amount of collagenase solution. (E) The remaining collagenase solution was added to the culture dish for mixing. After digestion at 37°C for 1 hour, at least two times the isolation medium was added to terminate the digestion, and the digested adipose tissue was blown until it was completely liquid. (F) After incubation with magnetic beads and antibodies, the SVF cell population was positively selected for Sca-1 in a sorter in a magnetic field.

[0089] Next, the microscopic morphology of ADSCs cultured by the three methods was observed and recorded. On the second day of the first medium exchange of ADSC-A, there were still a large number of lipid droplets, adipose tissue fragments and suspended cells, including hematopoietic cells, erythrocytes, adipocytes and irregularly shaped adherent cells ( Figure 3 A, Figure 3 G). In contrast, ADSC-M showed a significantly reduced cell number on day 2, indicating that Sca-1 negative cells were effectively eliminated ( Figure 3 D). By day 4, both ADSC-A and ADSC-M showed spindle-shaped features, but ADSC-M had less fat fragments and a more uniform morphology ( Figure 3 B, Figure 3E). On day 6, both groups reached 70-80% confluence, with spindle cells arranged in a distinct whorl, but ADSC-M had a higher density ( Figure 3 C, Figure 3 F). d After ADSC-AM was sorted by Sca-1 at the third generation, it showed a more three-dimensional spindle shape on the second day, with a uniform morphology and no obvious fat fragments ( Figure 3 H, Figure 3 I). On the 4th day, ADSC-AM reached 80-90% confluence and arranged in a whorl shape.

[0090] In summary, the results showed that: (1) ADSC-M and ADSC-AM had similar methods but different sequences, and compared with ADSC-A, they added magnetic bead sorting, which enabled the ADSC-AM method to enrich the cell population using Sca-1 as a marker of adipose-derived mesenchymal stem cells; (2) The culture time of the three methods was comparable, generally requiring three generations; (3) ADSC-A and ADSC-M cells had shorter and rounder morphologies, while ADSC-AM cells had thinner and more uniform morphologies and were able to quickly form vortices.

[0091] Figure 3 In (A) and (G), lipid droplets, tissue fragments and suspended cells can still be seen under the microscope after the first medium change on the second day of ADSC-A. (B) On the fourth day of ADSC-A, the cell morphology gradually showed a more uniform spindle shape. (C) On the sixth day of ADSC-A, the spindle cells reached 70-80% confluence and showed obvious vortex shape, but fat fragments and miscellaneous cells still existed. (D) On the second day of ADSC-MA, the total number of cells was significantly less than that of ADSC-A, and some lipid droplets, tissue fragments and suspended cells were present. (E) On the fourth day of ADSC-MA, the cell morphology gradually showed a more uniform spindle shape. (F) On the sixth day of ADSC-MA, the spindle cells reached 70-80% confluence, and a small amount of fat fragments and miscellaneous cells were still present. (H) On the second day of ADSC-MA inoculation, the cell morphology was relatively three-dimensional, slender and uniform. At this time, the background was clean and no lipid droplets and tissue fragments were seen. (I) On the fourth day of ADSC-MA, the confluence reached 80-90%, and the cells were arranged in a vortex shape.

[0092] 2. ADSC-AM has medium yield but the highest purity

[0093] To compare the cell yields obtained by the three purification methods, the same amount of adipose tissue from the bilateral inguinal fat pads of 4-week-old mice was used, and the cell counts of each generation were recorded (Table 1). The results showed that in most generations, the number of cells produced by ADSC-A was always the highest. After Sca-1 positive selection, the cell number of ADSC-M decreased by more than 50%, and the overall yield was the lowest. The cell counts of ADSC-AM in the 2nd and 3rd generations were similar to those of ADSC-A. After Sca-1 screening, the cell counts decreased by 30%, which was slightly higher than ADSC-M, but significantly higher than ADSC-M in subsequent passages.

[0094] In summary, the results showed that: (1) ADSC-A cells were the most abundant, but the purity might be lower; (2) ADSC-AM had a higher cell yield compared with ADSC-M, which might be related to the fact that some competitive hybrid cells were initially removed through adherent culture in the early stage of ADSC, thus forming the growth advantage of ADSC.

[0095] Table 1. Comparison of the yield of adipose-derived mesenchymal stem cells by three methods

[0096]

[0097] Note: Starting from the second generation, the baseline cell numbers of the three methods are similar, namely: ADSC-A, 2.26±0.11; ADSC-M, 2.03±0.10; ADSC-AM, 2.30±013.

[0098] 3. Enhanced proliferation activity of ADSC-AM

[0099] In order to evaluate the proliferation activity of ADSCs obtained by the three methods, a cell counting kit (CCK-8) was used to detect the proliferation of the third generation ADSC-A, ADSC-M and ADSC-AM within 6 days ( Figure 4 A). The results showed that the optical density (OD) value of ADSC-A was the lowest, indicating that the cells were aging. On the third day, the OD value of ADSC-AM was significantly higher than that of the ADSC-M group and the ADSC-A group, and the cell proliferation slope was also higher than that of the latter two groups. In addition, we used the fibroblast colony forming unit (CFU-F) experiment to detect the proliferation activity of the third generation ADSC-A, ADSC-M and ADSC-AM after 14 days ( Figure 3 C and D), which confirmed the results of CCK-8. After 14 days of culture at the same density, the proliferation activity of ADSC-AM was significantly higher than that of the other two groups, with the highest colony density and colony number (*P<0.05, **P<0.01, ***P<0.001).

[0100] In conclusion, the results indicated that ADSC-AM had stronger proliferation activity than ADSC-A and ADSC-M.

[0101] in, Figure 4 (A) Growth curve determined by CCK-8 method. (B) CFU-F count (n=3). (C) Crystal violet staining of cell colonies. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. *P<0.05, **P<0.01.

[0102] 4. ADSC-AM has a purer phenotype and a higher Sca-1 positive rate

[0103] In order to identify and compare the phenotypes of ADSCs obtained by the three methods, flow cytometry analysis was performed on the 4th generation ADSCs. Commonly used mouse ADSCs positive markers: CD44 and CD29, as well as endothelial cell marker CD31 and hematopoietic stem cell marker CD45 were selected. The flow cytometry results showed that the positive rate of CD44 was more than 90% in all three purification methods ( Figure 5 C). The positive rates of CD29 in ADSC-A and ADSC-M were both over 80%, while the positive rate of ADSC-AM was over 90% (Figure SD). In addition, the positive rates of CD31 and CD45 were both less than 5% ( Figure 5 A and 5B). After the ADSCs obtained by the three methods were cultured to the third generation, the cells were labeled with Sca-1 flow cytometry antibodies to detect their Sca-1+ positive rate. The results showed that the positive rate of Sca-1 was the lowest in ADSC-A, while the positive rate in ADSC-M increased, but still below 80%. In contrast, the positive rate of ADSC-AM was the highest, exceeding 90% ( Figure 5 E).

[0104] In conclusion, ADSC-AMs have a purer phenotype and a higher Sca-1 positivity rate compared with other methods.

[0105] in, Figure 5 In (AE), the cell markers CD31 (A) and CD45 (B) were negatively expressed in each group, and the cell markers CD44 (C), CD29 (D), and Sca-1 (E) were positively expressed (n=3). The data were analyzed by one-way ANOVA and expressed as mean ± standard error (M±SEM). ns, not significant; *p<0.05, **p<0.01, ***p<0.001.

[0106] 5. ADSC-AM exhibits stronger adipogenic differentiation potential

[0107] To evaluate the trilineage differentiation potential of the third generation ADSCs, osteogenic, adipogenic, and chondrogenic differentiation experiments were performed. All groups showed successful osteogenic (Alizarin red staining), adipogenic (Oil Red O staining), and chondrogenic (Alcian blue staining) differentiation ( Figure 6A. 6B and 6C). It is noteworthy that, although the positive areas of calcium nodules and mucopolysaccharide were not significantly different among the three groups, amADSCs showed a larger positive area of ​​lipid droplets during adipogenic differentiation.

[0108] In conclusion, all three types of ADSCs can differentiate into osteoblasts, adipocytes, and chondrocytes, but ADSC-AM has a stronger adipogenic differentiation ability.

[0109] in, Figure 6 In the figure, (A) Alizarin Red S staining after osteogenic induction, the histogram shows the quantitative analysis of calcium nodule area. (n=5) (B) Oil Red O staining after adipogenic induction, the histogram shows the quantitative analysis of lipid droplet area. (n=5) (C) Alcian Blue staining after chondrogenic induction, the histogram shows the quantitative sorting of mucopolysaccharide area. (n=5). Data were analyzed by one-way ANOVA and expressed as mean±SEM; ns, not significant; *P<0.05, **P<0.01, ***P<0.001.

[0110] 6. Comparison of ADSC purification methods

[0111] Table 2 summarizes the advantages and disadvantages of various purification methods (Table 2). ADSC-a had the highest cell count but lower purity. ADSC-M improved cell purity but significantly reduced cell yield. ADSC-AM had the highest medium yield and Sca-1+ cell ratio. ADSC-A and ADSC-AM were easily contaminated by cell debris and lipid droplets in the early stages of culture. Cell proliferation experiments (CCK-8 and CFU-F) showed that ADSC-A grew rapidly in the early stages of passage, and the growth rate slowed down when passaged to the third and fourth generations, and the cells became larger and aged. In contrast, ADSC-AM had the strongest proliferation ability.

[0112] In conclusion, ADSC-AMs stand out for their superior adipogenic differentiation potential and overall balance between yield and purity.

[0113] Table 2. Comparison of characteristics and functions of ADSCs obtained by three methods

[0114]

[0115] Conclusion

[0116] We have innovatively developed Sca-1 as a surface marker for a high-quality subpopulation of adipose mesenchymal cells, and developed a new method for efficiently enriching Sca-1-positive adipose-derived mesenchymal stem cells. The cell population obtained using this method has better characteristics of adipose-derived mesenchymal stem cells, namely, stronger proliferation activity and clone-forming ability, as well as tri-lineage differentiation capabilities of adipogenesis, osteoblastogenesis, and chondrogenesis.

[0117] Embodiment 2:

[0118] refer to Figure 7 , based on the above-mentioned embodiment 1, an application of efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells is disclosed.

[0119] The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells in Example 1 was applied to human adipose tissue to separate human adipose Sca-1+ mesenchymal stem cells, and Sca-1 positive adipose-derived mesenchymal stem cells with excellent performance were also obtained ( Figure 7 : Human Sca-1 positive adipose-derived mesenchymal stem cells were obtained by ADSC-AM method).

[0120] Among them, human body fat is mainly the fat under the skin of the abdominal wall, buttocks or thighs, specifically the subjects with informed consent, liposuction is performed on volunteers after informed consent in plastic surgery or medical beauty centers, or the fat obtained by liposuction during weight loss surgery is used.

[0121] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, characterized in that: Here are the steps: Choose the appropriate animal mouse; Isolation of primary ADSCs from mice; The stromal vascular fraction was cultured adherently, and Sca-1+ADSCs were separated by magnetic bead sorting to form ADSC-AM cells; The positive rate in ADSC-AM was detected by flow cytometry; The proliferation activity, osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation abilities of ADSC-AM cells were detected.

2. The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claim 1, characterized in that: The steps of isolating primary ADSCs from mice are as follows: Euthanasia was performed by carbon dioxide asphyxiation and immersion in 70% ethanol for 3 min; Inguinal fat was obtained and transferred to a 60 mm culture dish; After washing with PBS, the tissue was minced into 1-2 mm^3 fragments and digested in a solution containing 0.25% type II collagenase and 0.1% type I collagenase at 37°C for 1 h; The obtained SVF cell population was incubated with magnetic beads and antibodies, and then Sca-1 positive selection was performed in a sorter in a magnetic field.

3. The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claim 1, characterized in that: The steps of performing the adherent culture of ADSCs and separating Sca-1+ADSCs by magnetic bead sorting are as follows: SVF cells were directly cultured on the wall; SVFs were adherently cultured to the third generation, and +ADSCs were isolated by magnetic bead sorting. After purification, the culture medium was replaced every 2 days and cells were passaged at 80% confluence.

4. The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claim 1, characterized in that: The steps of detecting the positive rate in ADSC-AM by flow cytometry are as follows: ADSC-AM cells were dark stained with fluorescent antibodies for 45 min; After dark staining, the cells were washed with a buffer containing 0.5% BSA and detected by flow cytometry.

5. The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claim 1, characterized in that: The steps of detecting the proliferation activity, osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation ability of ADSC-AM cells are as follows: The proliferation activity of ADSC-AM cells was detected by cell counting reagent; The osteogenic differentiation ability of ADSC-AM cells was detected by osteogenic differentiation reagent; Adipogenic differentiation induction reagent was used to detect osteogenic and adipogenic differentiation in ADSC-AM cells; The chondrogenic differentiation ability of ADSC-AM cells was detected by chondrogenic differentiation reagent.

6. The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claim 5, characterized in that: The cell counting kit is used to detect the proliferation activity of ADSC-AM cells, and the steps are as follows: The third generation ADSC-AM cells were seeded in the well plate and cultured for 14 days; The cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for 20 min, and CFU-Fs were recorded and counted.

7. An application for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells, characterized in that: The method for efficiently enriching Sca-1 positive adipose-derived mesenchymal stem cells according to claims 1 to 6 is applied to human adipose tissue.

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