Preparation of human-derived induced pluripotent stem cell iPS-clone 12c and exosome and application of human-derived induced pluripotent stem cell iPS-clone 12c to hair growth
The exosomes of iPS-clone 12c inducible pluripotent stem cells promote the proliferation and differentiation of hair follicle stem cells, solve the limitations and side effects of existing hair loss treatment methods, and achieve efficient and low-side effects hair growth effects, which are more effective. Patients with hair loss have new treatment options.
Patent Information
- Application Number
- CN202411072526.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2024-12-13
AI Technical Summary
Existing hair loss treatments, such as drug treatment and hair transplantation, have limitations and side effects, which cannot effectively solve the problem of multiple types of hair loss, and are not sufficient for patients with large-scale hair loss to meet their needs.
By screening and culturing human-induced pluripotent stem cells iPS-clone 12c, its exosomes are prepared, and locally administered to promote the proliferation and differentiation of hair follicle stem cells and shorten the hair regeneration cycle, providing a new, low-side effects treatment with low side effects choose.
It significantly improves the hair growth effect, reduces inflammation, provides a high-safety and low-side effects treatment plan, is suitable for patients with various types of hair loss, and has a wide range of clinical application prospects.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a human induced pluripotent stem cell iPS-clone 12c, the preparation of exosomes and their application in hair growth. Background Art
[0002] With the aging of the population and changes in lifestyle, hair loss has become a global health and aesthetic challenge. Hair loss can be clinically divided into androgenic alopecia, alopecia areata, telogen effluvium, etc. Hair loss is a complex physiological process, and its causes involve genetics, hormone levels, age, environmental factors, lifestyle and other aspects. The causes of different types of hair loss are different, but they are all caused by hair follicle atrophy and degeneration. The hair growth cycle includes the growth phase, the regression phase and the resting phase. Generally, after the growth phase, the hair enters the regression phase and the resting phase, followed by physiological shedding. When the number of hairs lost per day exceeds 100, the dynamic balance of hair growth is broken, leading to pathological hair loss.
[0003] As the main treatments for hair loss, drug therapy and hair transplantation have significant limitations and problems. The commonly used drugs for the clinical treatment of hair loss are minoxidil, finasteride, dutasteride, flutamide, bicalutamide, spironolactone, etc. However, drug treatment of hair loss is usually only suitable for specific types of hair loss and requires long-term continuous use. Once the drug is stopped, the hair loss problem often reappears, causing patients to receive long-term drug treatment and may cause a series of side effects, such as scalp irritation and itching. In addition, the effect of drug treatment varies from individual to individual, and for some patients, the effect may not be significant. Although hair transplantation is a more direct treatment method, it also has many problems. First, as a surgical procedure, hair transplantation has certain surgical risks, including infection and bleeding, and requires a certain recovery period after surgery, which may affect the patient's daily life. Second, hair transplantation relies on the patient's own donor hair follicles. For patients with large areas of hair loss, the donor hair follicles may not be enough to meet the needs. Third, although the physician will try to imitate naturally grown hair during surgery, the transplantation effect may still be different from the natural state. Finally, the cost of hair transplantation is relatively expensive, which places a heavy financial burden on patients.
[0004] As an emerging regenerative medicine technology, stem cell therapy has shown great potential in the treatment of various diseases in recent years. Stem cells are special cells with the potential for self-proliferation and multidirectional differentiation. They can use the exosomes they secrete to regulate the growth of hair follicle stem cells, thereby promoting hair regeneration. Stem cell exosomes are nanoscale extracellular vesicles secreted by stem cells, containing a variety of bioactive molecules such as proteins, RNA, lipids, etc., which can promote the repair and regeneration of hair follicle stem cells by regulating intercellular communication and signal transduction.
[0005] Compared with the side effects and limitations of drug therapy and hair follicle transplantation, the advantages of stem cell exosomes in treating hair loss are: (1) Applicability to multiple types of hair loss. Stem cell exosomes contain a variety of biological molecules, such as proteins, RNA, lipids, etc. These biological molecules can regulate multiple physiological processes such as cell proliferation, differentiation, and immune regulation, and play a role in multiple hair loss mechanisms. Therefore, exosome therapy has a wide range of applicability and can be used for multiple types of hair loss, providing options for more patients. (2) No surgical risk. Compared with hair transplant surgery, exosomes do not require surgery for treating hair loss. They can be applied topically or injected locally into the scalp, avoiding surgical risks and postoperative discomfort. (3) Safety and patient-friendliness. Exosomes use natural biological signal transduction pathways, which are more in line with the physiological characteristics of the human body. They are expected to reduce the incidence of adverse reactions and improve the safety of treatment. They are a safer and more comfortable treatment option. Summary of the invention
[0006] Based on the problems existing in the background technology, the purpose of the present invention is to improve the biological activity and stability of stem cell exosomes and improve their therapeutic effect. A human induced pluripotent stem cell iPS-clone 12c, the preparation of exosomes and their hair growth application are proposed to provide a new, highly effective and low-side effect treatment option for hair loss patients.
[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A human induced pluripotent stem cell iPS-clone 12c, characterized in that: human induced pluripotent stem cell iPSC (ATCC cell line number: BYS0112) is plated in a 96-well plate by a limiting dilution method, placed in a hypoxic incubator for culture, and the feeder-free maintenance culture medium is replaced every day. The cell clones grown in the 96-well plate are continuously passaged for 30 times, and cell clones that have rapid growth, clear edges, good compactness, no differentiation characteristics, and uniform cobblestone morphology after 30 continuous passages under hypoxic conditions are screened out. The cell clone is iPS-clone 12c, and the cell clone is classified and named as human induced pluripotent stem cell iPS-clone 12c Homo sapiens. It was deposited in the China Center for Type Culture Collection on July 23, 2024, and the deposit number is CCTCC NO: C2024238.
[0008] Furthermore, the culture method is to digest the iPS-clone 12c clones and continue to expand and passage them in a feeder-free maintenance medium. When the cell confluence reaches 70% - 80%, the culture supernatant is collected and the iPS-clone12c cells are digested and passaged to ensure their continued growth and stability.
[0009] A method for preparing exosomes from human induced pluripotent stem cells iPS-clone 12c, characterized in that: S1: Screening and obtaining the human induced pluripotent stem cell line iPS-clone 12c; S2: Collect the culture supernatant of iPS-clone 12c, extract and purify exosome particles; S3: The obtained exosomes were subjected to morphological and compositional functional analysis to ensure the quality of the exosomes and their functional properties.
[0010] Furthermore, after removing subcellular components by high-speed centrifugation, the exosome precipitate is separated by ultra-high-speed centrifugation, and the exosome precipitate is resuspended and washed with PBS buffer, and then subjected to ultra-high-speed centrifugation to obtain exosomes with higher purity.
[0011] Furthermore, density gradient centrifugation or immunocapture technology is used to further purify exosomes to ensure their purity and functionality.
[0012] Furthermore, transmission electron microscopy, nanoparticle tracking analysis, western blotting, and proteomics were used to analyze the morphology and compositional functions of the obtained exosomes.
[0013] An application of exosomes from human induced pluripotent stem cell iPS-clone 12c, characterized in that: the application promotes hair regeneration, including improving hair follicle atrophy, repairing hair follicle damage, and promoting hair follicle regeneration.
[0014] A method for applying human induced pluripotent stem cell iPS-clone 12c exosomes, characterized in that the iPS-clone 12c exosomes are locally applied to a site where hair regeneration needs to be promoted, thereby stimulating the proliferation and differentiation of hair follicle stem cells and shortening the hair regeneration cycle.
[0015] Furthermore, the local administration method is one or more of injection, spray, patch, local immersion, and nanocarrier delivery.
[0016] The following beneficial effects can be obtained through the above technical solution: The cell source is safe and reliable without any ethical controversy: iPS-clone 12c is derived from human induced pluripotent stem cells provided by ATCC. After strict culture and screening, the cell line has shown strong vitality, high stability and no differentiated phenotype after 30 consecutive passages under hypoxic conditions. It is easy to expand and passage, and has no immune rejection problems, making it suitable for clinical research and application.
[0017] iPS-clone 12c exosomes have the characteristics of promoting hair growth and reducing inflammation: iPS-clone 12c exosomes can directly act on hair follicle stem cells, promote the proliferation of hair follicle stem cells, help to improve the regulation of hair follicle stem cells from the root, and are suitable for patients with various types of hair loss.
[0018] In summary, the present invention not only shows significant effects in promoting hair growth, but also has the advantages of high safety and low side effects. It provides a high-efficiency, low-side effect treatment option for patients with hair loss and shows broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the morphology of iPS-clone 12c exosomes observed under electron microscopy technology.
[0020] Figure 2 Figure 2 shows the particle size distribution of exosomes from iPS-clone 12c.
[0021] Figure 3 This is a Western blotting image of the exosome marker proteins of iPS-clone 12c.
[0022] Figure 4 This is a diagram based on Gene Ontology (GO) data analysis.
[0023] Figure 5 This is a diagram of DIA proteomics differential analysis.
[0024] Figure 6 This is a diagram of hair growth in the mouse experiment of Example 4 of the present invention.
[0025] Figure 7 These are photos of the hair loss area before and after medication in the clinical trial (volunteers) of Example 5 of the present invention. DETAILED DESCRIPTION
[0026] The following are examples and attached Figure 1-7 The present invention is further described as follows: The present invention constructs an induced pluripotent stem cell line iPS-clone 12c obtained by screening, and the cell clone classification is named human induced pluripotent stem cell iPS-clone 12c Homo sapiens, which was deposited in China Center for Type Culture Collection on July 23, 2024, with a deposit number of CCTCC NO: C2024238, and a deposit address: China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and provides a method for preparing iPS-clone 12c exosomes and an application thereof for hair regeneration, comprising the following steps: Construction and culture of iPS-clone 12c: Human induced pluripotent stem cells (iPSCs) purchased from the American type culture collection (ATCC) (ATCC cell line number: BYS0112) were revived and plated in 96-well plates by limiting dilution and placed in a hypoxic (5% O2) incubator for culture. The feeder-free maintenance medium was changed every day. The cell clones grown in the 96-well plates were passaged 30 times in succession, and the cell clones that still had rapid growth, clear edges, good compactness, no differentiation characteristics, and uniform cobblestone morphology after 30 consecutive passages under hypoxic conditions were selected and named iPS-clone12c. The iPS-clone 12c clones were digested and continued to be expanded and passaged in feeder-free maintenance medium. When the cell confluence reached 70% - 80%, the culture supernatant was collected, and the iPS-clone 12c cells were digested and passaged to ensure their continued growth and stability.
[0027] Extraction and purification of exosomes: The culture medium of iPS-clone 12c cells was collected and placed under low-speed centrifugation conditions (2000 g, 4°C, centrifugation for 10 min), the cell pellet was discarded, and the supernatant was collected and filtered through a 0.22 μm filter.
[0028] The supernatant obtained by the above centrifugation is further processed. Method 1 can remove subcellular components such as cell debris by high-speed centrifugation (10,000g, 4°C, centrifugation for 30 minutes), and then separate the exosome precipitate by ultra-high-speed centrifugation (100,000g, 4°C, centrifugation for 70 minutes). The exosome precipitate is resuspended and washed with PBS buffer, and then subjected to ultra-high-speed centrifugation (100,000g, 4°C, centrifugation for 70 minutes) to obtain exosomes with higher purity.
[0029] Method 2 can use density gradient centrifugation or immunocapture technology to further purify exosomes to ensure their purity and functionality.
[0030] Identification and Characterization of Exosomes: Transmission electron microscopy (TEM): used to observe the morphology of exosomes to ensure that they have a typical double-layer membrane structure and uniform particle size.
[0031] Nanoparticle Tracking Analysis (NTA): Determine the particle size distribution of exosomes to ensure that their particle size is mainly concentrated between 30-150nm and there is a single particle size peak.
[0032] Western Blot: Detect specific marker proteins (CD63, CD81, CD9, TSG101, etc.) in exosomes to ensure the quality and function of exosomes.
[0033] DIA relative quantitative exosome proteomics: High-throughput analysis and identification of proteins in exosomes helps to reveal the complex composition and functional mechanism of exosomes, and also provides important scientific basis for their use as biomarkers and therapeutic carriers.
[0034] An application of exosomes from human induced pluripotent stem cell iPS-clone 12c to promote hair regeneration, including improving hair follicle atrophy, repairing hair follicle damage, and promoting hair follicle regeneration.
[0035] The iPS-clone 12c exosomes are locally applied to the part where hair regeneration needs to be promoted to stimulate the proliferation and differentiation of hair follicle stem cells and shorten the hair regeneration cycle. The local application method is one or more of injection, spray, patch, local immersion, and nanocarrier delivery.
[0036] Example 1: Construction of iPS-clone 12c cell line and preparation of exosomes Human induced pluripotent stem cells (ATCC cell line number: BYS0112) purchased from ATCC were revived, and iPSC cells were seeded in 96-well plates coated with matrigel (Nucleus Biologics, Vitronectin XF™) by limiting dilution method, and placed in a hypoxic (5% O2) incubator for culture, using a feeder-free maintenance medium designed for iPSC (StemCell, mTeSR™1), and the medium was changed every day. The cell clones grown in the 96-well plate were passaged 30 times in succession, and the morphological characteristics of iPSC were observed under a microscope. The cell clones that had rapid growth, clear edges, good compactness, no differentiation characteristics, and uniform cobblestone morphology after 30 consecutive passages under hypoxic conditions were selected and named iPS-clone 12c. Then, the iPS-clone 12c clones were digested using a cell dissociation reagent (StemCell, ReLeSR™) and transferred to a new culture dish coated with Matrigel, recorded as the first generation (P1), and expanded and passaged in a feeder-free maintenance medium. The iPS-clone 12c obtained by screening was cultured in a feeder-free maintenance medium, and the medium was changed every day. When the cell confluence reached 80%, the culture supernatant was collected, and the iPS-clone 12c cells were digested and passaged.
[0037] According to the above method, the supernatant of iPS-clone 12c cells was collected, and exosomes were extracted from the cell culture supernatant using ultracentrifugation. First, centrifuge at 2,000g for 10 minutes to remove cells and collect the supernatant. Then, centrifuge at 10,000g for 30 minutes to remove cell debris and other subcellular components, and collect the supernatant again. Finally, centrifuge at 100,000g for 70 minutes to obtain exosome pellets. The pellet was resuspended in PBS and ultracentrifuged again at 100,000g for 70 minutes to obtain highly pure exosomes. The iPS-clone 12c exosomes resuspended in PBS were sterilized by filtration through a 0.22μm filter membrane, aliquoted and stored at -80°C for later use.
[0038] Example 2: Identification and characterization of exosomes 2.1 Transmission electron microscopy observation of the morphology of iPS-clone 12c exosomes The morphology of exosomes was observed by transmission electron microscopy (TEM). A small amount of the iPS-clone 12c exosome suspension obtained in Example 1 was dropped on the surface of the copper mesh, left to stand for 10 minutes, stained with 2% phosphotungstic acid for 1 minute, and observed under an electron microscope after drying. Figure 1 The typical double-layer membrane structure of iPS-clone 12c and its uniform size are shown.
[0039] 2.2 Exosome particle size analysis The exosome samples were analyzed for particle size using nanoparticle tracking analysis (NTA) technology. The iPS-clone 12c exosome suspension obtained in Example 1 was diluted to an appropriate concentration (usually diluted to 108-109 particles / mL). A calibrated NanoSight NS300 nanoparticle tracking analyzer (Malvern Panalytical, UK) was used for at least 3 independent measurements, each lasting 60 seconds to ensure the repeatability and accuracy of the results. After each measurement, the sample pool was washed with PBS to prevent cross contamination. The captured video was analyzed using NTA software to calculate the particle size distribution and concentration of the exosome particles. The diameter of the particles was determined by the Brownian motion trajectory, and the particle size distribution was plotted. Figure 2 The results showed that the particle size of iPS-clone 12c was mainly concentrated between 30-150 nm, and there was a single peak, indicating that the particles in the solution were relatively uniform.
[0040] 2.3 Detection of exosome marker proteins Western Blot was used to detect the expression of specific marker proteins in iPS-clone 12c exosomes. Proteins in exosomes were extracted, separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes. Anti-CD81, CD9, and TSG101 antibodies were used for incubation, followed by development with corresponding secondary antibodies. Figure 3 The development results of exosome marker proteins CD81, CD9, and TSG101 were shown, demonstrating that the extracted iPS-clone 12c exosomes have typical protein biomarkers.
[0041] Example 3: DIA proteomic detection of exosomes The whole proteome of iPS-clone 12c exosomes and unscreened normal iPSC exosomes was analyzed by DIA relative quantitative proteomics analysis. Figure 4 Based on the Gene Ontology (GO) database, the GO functional annotation and enrichment of iPS-clone 12c exosome proteins in three branches: biological process (BP), cellular component (CC) and molecular function (MF) were shown. Figure 5As shown in the figure, differential proteins were screened and analyzed between iPS-clone 12c exosomes and normal iPSC exosomes, and a total of 702 differentially upregulated proteins in iPS-clone 12c exosomes were screened. Based on the GO database, GO functional annotation and enrichment analysis of the screened differential proteins were performed in three branches: biological process, cellular component and molecular function. It was found that compared with normal iPSC exosomes, biological processes such as protein transport, positive regulation of exosome secretion, and positive regulation of cell growth in iPS-clone 12c exosomes were significantly upregulated; its cellular components such as membrane, exosomes, and plasma membrane were significantly upregulated; its molecular functions such as protein binding, ATP binding, and cadherin binding were significantly upregulated. At the same time, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to annotate the differentially expressed proteins, and it was found that compared with normal iPSC exosomes, iPS-clone 12c exosomes showed significant upregulation in pathways related to exosome synthesis and secretion and pathways related to regulating cell growth cycle (mTOR signaling pathway, PI3K-Akt signaling pathway, and TGF-β signaling pathway, etc.). The above results show that at the level of whole protein analysis comparison, compared with unscreened normal iPSC, iPS-clone 12c cell line has stronger exosome secretion ability and the ability to regulate the growth cycle of hair follicle cells.
[0042] Example 4: Mouse hair regeneration experiment The androgenic alopecia model mice were randomly divided into two groups after hair removal. One group was smeared with saline as the control group, and the other group was smeared with iPS-clone 12c exosome suspension. The application was done once a day for 2 consecutive weeks. The hair regeneration of the mice was observed and recorded. Figure 6 The changes in mouse hair growth and the coverage rate of new hair in the hair loss area were demonstrated. The results showed that the hair regeneration of the mice in the experimental group treated with iPS-clone12c exosomes was significantly better than that in the control group.
[0043] Example 5: Clinical Trial (Volunteers) As attached Figure 7 As shown in the figure, the volunteers were significantly affected by androgenic alopecia on their heads before the trial. After 85 days of the trial, the areas where there was almost no hair growth before became densely covered with hair, and hair also grew in the central area. After the trial, the volunteers washed and cared for their hair normally, and the new hair was not damaged. By taking photos to record the changes in the hair in the affected area, it was found that the hair regeneration effect of the volunteers using iPS-clone 12c exosomes was very significant.
[0044] In summary: The present invention discloses a human induced pluripotent stem cell line iPS-clone 12c obtained by culture screening, which has the characteristics of rapid growth, clear edges, good compactness, non-differentiation characteristics and uniform cobblestone morphology. In addition, the present invention provides an application of iPS-clone 12c exosomes for hair regeneration. The exosomes prepared using the supernatant of the iPS-clone 12c culture solution as raw material have a significant effect in promoting hair regeneration, and are highly safe, have no obvious side effects, and have broad application prospects. The above embodiments are only preferred embodiments of the present invention. Those skilled in the art can make various improvements and changes to the above embodiments without departing from the principles and essence of the present invention, and these improvements and changes should also be regarded as the scope of protection of the present invention.
Claims
1. A human induced pluripotent stem cell iPS-clone 12c, characterized in that: The human induced pluripotent stem cell iPSC was plated in a 96-well plate by limiting dilution method and placed in a hypoxic incubator for culture. The feeder-free maintenance culture medium was replaced every day. The cell clones grown in the 96-well plate were serially passaged 30 times, and the cell clones with rapid growth, clear edges, good compactness, non-differentiation characteristics and uniform cobblestone morphology after 30 consecutive passages under hypoxic conditions were screened. The cell clone was human induced pluripotent stem cell iPS-clone 12c, which was deposited in the China Center for Type Culture Collection on July 23, 2024, with the deposit number CCTCC NO: C2024238.
2. The human induced pluripotent stem cell iPS-clone 12c according to claim 1, characterized in that: The culture method is to digest the iPS-clone 12c clones and continue to expand and passage them in a feeder-free maintenance medium. When the cell confluence reaches 70% - 80%, the culture supernatant is collected and the iPS-clone 12c cells are digested and passaged to ensure their continued growth and stability.
3. A method for preparing exosomes of human induced pluripotent stem cell iPS-clone 12c according to claim 1, characterized in that: S1: Screening and obtaining the human induced pluripotent stem cell line iPS-clone 12c; S2: Collect the culture supernatant of iPS-clone 12c, extract and purify exosome particles; S3: The obtained exosomes were subjected to morphological and compositional functional analysis to ensure the quality of the exosomes and their functional properties.
4. A method for preparing exosomes from human induced pluripotent stem cells iPS-clone 12c according to claim 3, characterized in that: After removing subcellular components by high-speed centrifugation, the exosome precipitate is separated by ultra-high-speed centrifugation, the exosome precipitate is resuspended and washed with PBS buffer, and then subjected to ultra-high-speed centrifugation to obtain exosomes with higher purity.
5. The method for preparing exosomes from human induced pluripotent stem cells iPS-clone 12c according to claim 3, characterized in that: Density gradient centrifugation or immunocapture techniques are used to further purify exosomes to ensure their purity and functionality.
6. The method for preparing exosomes from human induced pluripotent stem cells iPS-clone 12c according to claim 3, characterized in that: Transmission electron microscopy, nanoparticle tracking analysis, western blotting, and proteomics were used to analyze the morphology and composition of the obtained exosomes.
7. An application of exosomes from human induced pluripotent stem cells iPS-clone 12c, characterized in that: Applications that promote hair regeneration include improving hair follicle atrophy, repairing hair follicle damage, and promoting hair follicle regeneration.
8. An application of exosomes from human induced pluripotent stem cell iPS-clone 12c for hair growth, characterized by: By topically administering iPS-clone 12c exosomes to the area where hair regeneration is needed, the proliferation and differentiation of hair follicle stem cells are stimulated, shortening the hair regeneration cycle.
9. The application of exosomes from human induced pluripotent stem cell iPS-clone 12c in hair growth according to claim 8, characterized in that: The local administration method is one or more of injection, spray, patch, local immersion, and nanocarrier delivery.