WO2019148395A1 - Artificial hair follicle, preparation method therefor and use thereof - Google Patents

Artificial hair follicle, preparation method therefor and use thereof Download PDF

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WO2019148395A1
WO2019148395A1 PCT/CN2018/074827 CN2018074827W WO2019148395A1 WO 2019148395 A1 WO2019148395 A1 WO 2019148395A1 CN 2018074827 W CN2018074827 W CN 2018074827W WO 2019148395 A1 WO2019148395 A1 WO 2019148395A1
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stem cells
pluripotent stem
cells
skin
embryo
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PCT/CN2018/074827
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Chinese (zh)
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王旭升
吴耀炯
牟丽莎
曹楠
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深圳市祥云生物科技有限公司
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0625Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627Hair cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

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  • the present invention relates to the field of biotechnology, and in particular, to artificial hair follicles and methods for their preparation and use.
  • Hair loss caused by hair loss has become a sub-health problem that is plaguing modern males (including some females). Although hair loss does not cause serious physical health problems, hair loss can seriously affect an individual's psychological state, so it will be serious. Reduce the quality of life of individuals. According to incomplete statistics, there are currently more than 200 million people in China who have different degrees of hair loss. In the world, this group of people has exceeded 1 billion.
  • Pathological abnormal hair loss such as male baldness
  • the hair follicles are in a long-term rest period and cannot enter the growth cycle.
  • drugs for pathological alopecia and these drugs are only for very few.
  • the proportion of patients with effects, such as minoxidil and finasteride are the two most widely used anti-hair loss drugs in clinical practice. It is not only for long-term use, but also for different degrees of side effects. Relatively weak.
  • Stem cells are the initial source of the human body and its various tissue cells. Its most prominent biological characteristics are its ability to self-renew and proliferate, as well as the potential for multi-directional differentiation. Stem cells are classified into adult stem cells (emmatic stem cells) and embryonic stem cells (ES cells) according to different sources.
  • adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, and the like, which are present in adult tissues.
  • the Yamanaka team at Kyoto University in Japan used in vitro gene transfection technology to screen four transcription factors including Oct4, Sox2, c-Myc, and Klf4 from 24 factors, and the above four by retrovirus.
  • the transcription factor is introduced into embryonic mouse fibroblasts or adult mouse tail skin fibroblasts, and Fbx15+ pluripotent stem cell lines are obtained under the condition of mouse ES (embryonic stem cell) cells.
  • the cell morphology and proliferation of such iPS cells are obtained.
  • the ability, surface antigen marker, gene expression profile, epigenetic state of pluripotent stem cell-specific genes, and telomerase activity are similar to those of hES cells, and both in vitro culture and in teratoma formation in mice.
  • Different cell types can be differentiated into three germ layers (Takahashi K et al. Cell 2007; 131: 861-872). This major breakthrough, known as the “milestone” of the biological sciences, is expected to help scientists bypass the ethical and moral disputes of cloning technology and open the door to medical applications.
  • FUE follicular unit extraction
  • FUE has the advantage of wireless scarring, there are still other problems and potential long-term side effects. As with full-thickness skin incisions, harvesting each follicular unit produces a scar of a 1 mm diameter punch. These scars have an effect on the coloration and development of the remaining hair follicles. Furthermore, whether the FUE is performed by manual, motorized or robotic perforation, as with the donor elliptical scalp cutting harvesting hair follicles, there is a risk of significant consumption of hair from the donor area, ie the hair of the donor area will be reduced in detail. This may result in an iatrogenic "moth” or "pseudo-syphilis" appearance ( N et al.
  • FUE is an effective and useful way to get the donor's hair. It produces less visible scars than the elliptical scalp cut donor hair follicles. As with any surgical technique, this approach has limitations and side effects.
  • hair follicle transplantation has become an emerging solution to the problem of hair loss in recent years.
  • the method of hair follicle transplantation is to take the hair follicle of the posterior occipital region and then transplant the hair follicle to the same person's forehead hair.
  • the method is immediate and effective, but after all, it is like disassembling the east wall to make up the western wall. It is effective for patients with a small amount of hair loss, but it cannot have a good therapeutic effect for patients with large area hair loss.
  • hair follicle extraction is a mechanical damage to the hair follicle, affecting the survival rate of the hair follicle.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the present invention proposes a hair follicle regeneration method based on human autologous stem cells and heterogeneous vectors.
  • the invention proposes a method of obtaining artificial hair follicles.
  • the method comprises: transplanting human pluripotent stem cells into the skin of a heterologous embryo, the heterologous embryos being in a hair formation stage; and continuing to culture the embryos transplanted with the pluripotent stem cells in the xenogeneic mother, Until the embryo is delivered; and the hair follicle in the skin of the pluripotent stem cells is transplanted in the embryo to obtain the artificial hair follicle.
  • the artificial hair follicle obtained by the method according to the embodiment of the invention has a large number, and the structural morphology is similar to that of the hair follicle in normal skin, and can be used for hair follicle transplantation of different parts of the human body, such as scalp, eyebrow, beard, body hair, etc.; Until the pluripotent stem cell provider does not have immune rejection, the safety is greatly improved.
  • the above method may further include at least one of the following additional technical features:
  • the human pluripotent stem cells are induced pluripotent stem cells (iPSCs), and the transplanting further comprises: differentiating the induced pluripotent stem cells into skin-like organs, the skin-like organs including ectoderm Cells, mesoderm cells and neuroendodermal cells.
  • the stem cells first differentiate into three germ layers, and then further differentiate into skin-like organs, thereby further differentiating into hair follicle structures.
  • the differentiation of the induced pluripotent stem cells into a dermatophyte comprises: suspending the induced pluripotent stem cells to obtain a 3D cell sphere; and further culturing the 3D cell sphere so that Get skin-like organs.
  • the source of the induced pluripotent stem cells is not particularly limited and may be derived from any one of adult cells. According to an embodiment of the invention, the induced pluripotent stem cells are derived from blood cells or skin cells.
  • the differentiation of the induced pluripotent stem cells into a dermatophyte is carried out under conditions in which the pluripotent stem cells are contacted with BMP-4, SB431542, FGF-2 and LND.
  • the concentration of BMP-4 is 50 ng/mL. Further, the induction efficiency of the skin-like organs is further improved.
  • the transplanting further comprises: digesting the skin-like organ to obtain a single-cell mixture.
  • the single cell mixture is mixed with a matrigel or gel-like substance. Further, the single cell mixture can form a membranous cell layer under the induction of a factor that promotes hair follicle formation. According to an embodiment of the invention, the thickness of the membranous cell layer is the same as the thickness of the skin of the recipient xenogenic embryo.
  • the Matrigel comprises at least one selected from the group consisting of Geltrex and ColI.
  • the human pluripotent stem cells comprise stem cells selected from the group consisting of hair follicle stem cells, skin stem cells, and other hair follicle forming or inducing abilities.
  • the human pluripotent stem cells comprise genetically modified pluripotent stem cells or induced pluripotent stem cells.
  • the genetic modification is gene knockout or gene expression.
  • the transplanting further comprises: mixing the human pluripotent stem cells with a factor that promotes hair follicle formation. Furthermore, the hair follicle formation efficiency is greatly improved.
  • the factor that promotes hair follicle formation is a Wnt10b factor. Furthermore, the hair follicle formation efficiency is further improved.
  • the concentration of the Wnt10b factor is 1 ug/mL. Furthermore, the hair follicle formation efficiency is further improved.
  • the xenogeneic embryo comprises at least one selected from the group consisting of a pig embryo, a primate embryo, a rabbit embryo, a bovine embryo, and a sheep embryo.
  • the heterologous animal carrier must meet clinical hygiene standards when used, and can neither carry any clinically specified pathogens at risk of disease.
  • the heterogeneous embryo only provides an environment for the development of human stem cells into hair follicles, and finally it is necessary to completely remove all animal-derived tissues and cells when separating mature artificial hair follicles.
  • the separation is carried out within 0 to 6 months after the embryo is delivered.
  • the hair follicle at this time has a relatively complete structure, has a transplant condition and is highly regenerative.
  • the present invention further comprising, after transplanting the obtained artificial hair follicle back to the skin of the stem cell provider, determining whether the hair follicle is viable after transplantation and has a normal growth cycle. If the hair follicle is in a growth period of more than 3 months, and enters the growth period within 1-3 months after entering the rest period, it is judged that the hair follicle survives after transplantation and has a normal growth cycle. It can be used for subsequent hair follicle transplantation.
  • the invention proposes an artificial hair follicle.
  • the hair follicle is obtained by the method described above.
  • the artificial hair follicle according to the embodiment of the present invention can be peeled off from the entire skin tissue, and the damage of the hair follicle can be minimized; and the artificial hair follicle according to the embodiment of the present invention is an autologous hair follicle produced by the autologous stem cell, if these hair follicles are transplanted back Stem cell providers do not have immune rejection and are safer.
  • the invention proposes a method of hair follicle planting.
  • the method comprises inoculating the artificial hair follicles described above onto the surface of the autologous skin.
  • the method according to an embodiment of the present invention can be applied to hair follicle planting of male hair loss (sharp top), hair follicle planting for hair encryption, hair follicle planting for hairline adjustment, hair follicle planting of scar scalp, eyebrow planting, and beard planting. And body hair planting.
  • FIG. 1 is a flow chart of a hair follicle regeneration method based on autologous induced pluripotent stem cells and xenogeneic vectors according to an embodiment of the present invention
  • FIG. 2 is a diagram showing induced differentiation of iPS cells into skin cells according to an embodiment of the present invention
  • a mononuclear cells are isolated from the blood; b, mononuclear cells are induced into iPS cells, iPS cells form clones; c, iPS cells differentiate into skin precursor cells;
  • 3 is a mixture of differentiated iPS cells and a gel, and transplantation of cells according to an embodiment of the present invention
  • FIG. 4 is a diagram showing the results of formation of a hair follicle structure by stem cells after transplantation according to an embodiment of the present invention
  • Figure 5 is a graph showing the results of staining of hair follicles and skin structures formed entirely from human stem cells by human cell-specific marker MAB1281 staining in accordance with an embodiment of the present invention.
  • the invention provides a hair follicle regeneration method based on human autologous stem cells and a heterogeneous vector, wherein the method for regenerating hair follicles comprises the separation, culture and induction of human cells or stem cells, wherein there are two methods for obtaining stem cells, the first one is direct Separating hair follicle stem cells or skin cells from alopecia patients; the second is to induce adult cells (such as blood cells and skin cells) into pluripotent stem cells with multipotential differentiation potential, which is induced pluripotent stem cells (iPS cells). That is, to obtain a hair loss patient or an induced pluripotent stem cell requiring a hair transplanter, the technique has been effectively implemented.
  • iPS cells induced pluripotent stem cells
  • the induced pluripotent stem cells are obtained, they are first differentiated into ectodermal cells, mixed with Matrigel or gel-like substances, and factors that promote the differentiation and development of ectodermal cells to hair follicles are added, and the induced cells and gels are added.
  • the scaffold material forms a membranous cell layer.
  • the thickness of the membranous cell layer should be the same as the thickness of the fetal skin of the recipient xenobiotic.
  • the microenvironment is most suitable for hair follicle regeneration, so the human stem cell which has been differentiated into the ectoderm after transplantation will be effective under the action of hair follicle differentiation inducing factor.
  • hair follicle differentiation inducing factor Form hair follicles.
  • induction of ectodermal differentiation is not required prior to transplantation, but in order to promote the efficiency of hair follicle formation, it is still necessary to mix with factors that promote hair follicle formation before transplantation.
  • the human stem cells transplanted in the skin have partially developed into hair follicles.
  • the hair follicles have a relatively complete structure, and some hair follicles have hair growth.
  • the hair follicles already have the transplantation conditions, so the human hair follicles can be separated when the carrier animal fetus is born, and then planted.
  • the carrier animal fetus can also be raised for a period of time, allowing the human hair follicles carried by it to further mature and then planted separately.
  • Heterogeneous carrier animals include different breeds of pigs, primates and rabbits, cattle, and sheep.
  • pigs that achieve medical-grade hygiene should be the preferred animals. All use of heterologous animal carriers must meet clinical hygiene standards in practice, and should not carry any clinically specified pathogens at risk of disease. For pigs, strains that do not carry endogenous retroviruses are used.
  • the artificial hair follicles of the invention have many practical application directions, including hair follicle cultivation for treating male hair loss (Xieding), hair follicle cultivation for hair encryption, hair follicle cultivation for hairline adjustment, and scarring. Hair follicle cultivation of the scalp, including eyebrow planting, beard planting and body hair planting. Since the invention is characterized by a new hair follicle regenerated by autologous stem cells, it has several advantages in application. First, the post-cultured stem cells or induced pluripotent stem cells can be quantitatively expanded in a large amount.
  • the method can obtain any number of hair follicles; stem cells can be added with different growth factors in the development stage of the carrier animal, and the hair follicles can be controlled to grow into different sizes and shapes, thereby being used for hair follicle regeneration in different parts, such as eyebrows and beards;
  • the separation of the hair follicle peels the hair follicle from the entire skin tissue, thereby minimizing the damage to the hair follicle;
  • the regenerated hair follicle of the present invention is an autologous hair follicle produced by the autologous stem cell, so the transplantation of the hair follicle back to the stem cell provider is not An immune rejection will occur.
  • FIG. 1 a flow chart of a hair follicle regeneration method based on autologous induced pluripotent stem cells and xenogeneic vectors according to an embodiment of the present invention is referred to FIG.
  • MNC mononuclear
  • induced pluripotent stem cells are then used to produce induced pluripotent stem cells by following the steps below, placing 1-2 ⁇ 10 4 MNCs in one well of a 96-well plate and using the CytoTune-iPS reprogramming kit (DNAVEC, Tsukuba, Japan) Infected with OCT4, SOX2, KLF4, cMYC F-deficient Sendai virus vector (Sev/ ⁇ F), each of which has a multiplicity of infection (MOI) of 5-10.
  • MOI multiplicity of infection
  • the medium was changed to human ES (human embryonic stem cell) medium supplemented with 8 ng/ml bFGF, and changed daily with fresh medium. Morphology similar to ES cloning began to appear around day 13 post infection; clones were picked on day 21 or 28, expanded, and pluripotency markers were examined by immunofluorescence or flow cytometry.
  • the method for detecting pluripotency markers by immunofluorescence was to wash iPS cells three times with D-PBS and fix them in 4% paraformaldehyde for 10 minutes at room temperature. After washing with D-PBS, the cells were ruptured with methanol (room temperature for 1 minute), washed with D-PBS and blocked in PBS-5% sputum serum for 30 minutes.
  • Anti-Oct4 (mouse monoclonal), Tra1-81 (mouse monoclonal) and Nanog antibodies were incubated overnight at 4 °C.
  • the cells were washed 3 times with D-PBS and subjected to a suitable fluorescent secondary antibody (Invitrogen) for 1 hour at room temperature, followed by DAPI staining for 1 minute.
  • a suitable fluorescent secondary antibody (Invitrogen) for 1 hour at room temperature, followed by DAPI staining for 1 minute.
  • the expression of pluripotency markers was examined under a fluorescence microscope.
  • the cells were washed again with D-PBS, resuspended in 500 ⁇ l of human ES cell culture medium and injected subcutaneously into SCID mice (Taconic). After 4-6 weeks of injection, tumors were removed from euthanized mice and fixed in 4% paraformaldehyde. The samples were embedded in paraffin and sectioned for analysis according to hematoxylin and eosin staining.
  • the iPSC clones were treated with 1 mg/ml collagenase IV (Invitrogen) for 10 minutes, washed with D-PBS, and dissociated into agglomerates by scraping and pipetting several times.
  • the iPSC blocks were coated onto Lipidure coated plates (NOF Corporation, Irvine, CA, USA) in ES medium without bFGF. Change the medium once a day. After 8 days of suspension culture, embryoid bodies (EB) were transferred to gelatin-coated plates and cultured for an additional 8-10 days in the same medium. The expression of the cell marker is then analyzed.
  • iPSC differentiation into skin-like organs the inventors passaged 1:8 of iPSCs. Preheat with N2B27 medium and Dispase in a 37 ° C water bath. The number of clones was confirmed using a microscope. The medium was gently aspirated from the Petri dish.
  • the cell clones are very loosely attached to the surface of the culture dish and may peel off if too much force is used.
  • Gently add 2 mL of pure DMEM/F12. Repeat the washing 3 times. 2 mL of N2B27 complete medium was added to the Petri dish, and the colonies were gently scraped from the culture plate. The cells were transferred from the culture dish to a 15 mL centrifuge tube, and 6 mL of N2B27 complete medium was added to bring the total volume of the cell suspension to 8 mL. Gently mix the cell suspension to break up large cells. Transfer 1 mL of the cell suspension to the prepared coated petri dish. Transfer the newly plated cells to the incubator, gently rock the plate back and forth and evenly distribute it. The cells were incubated overnight in a 37 ° C tissue incubator.
  • iPSCs Induction of iPSC cells into 3 germ layer cells and subculture is performed in a biosafety cabinet. The state of the cells was observed on the second day after passage to confirm the adherence of iPSC. If iPSCs begin to form clones, the following differentiation experiments will continue. Initially, the day of differentiation was day 0. On day 0, iPSCs were dissociated with 1X TrypLE Express enzyme (GIBCO) or Accutase cell dissociation reagent (GIBCO), resuspended in ectodermal differentiation medium, and seeded in 96 wells. On a low cell adhesion U-bottom plate, 100 ⁇ L per well, 3 ⁇ 10 3 cells per well.
  • GEBCO TrypLE Express enzyme
  • GEBCO Accutase cell dissociation reagent
  • the working concentration of BMP-4 was increased to a final concentration of 50 ng/mL, which improved the skin-like organ induction efficiency in quality.
  • 25 ⁇ L of fresh ectodermal differentiation medium (without Matrigel) containing 6 ⁇ M LDN (Stemgent) and 150 ng/mL FGF-2 (PeproTech) was added to each well to a final concentration of 1 ⁇ M LDN and 25 ng/mL FGF- 2.
  • the final volume of the culture solution was 150 ⁇ L/well.
  • each cell aggregate was transferred to a single well on a 24-well low cell adhesion plate (Nunclon Sphera) in 500 [mu]L of maturation medium containing 1% (v/v) Matrigel. Starting from day 10, half of the medium (250 ⁇ L) was removed every other day and supplemented with 250 ⁇ L of fresh mature medium (without Matrigel) until day 30.
  • the hair follicle structure is formed by using induced pluripotent stem cells, and the specific process is as follows: (1) The partially differentiated induced pluripotent stem cells obtained in Example 2 are cultured, and after being digested with Accutase, a cell suspension having a total cell count of 2 million is prepared. Centrifuge at 300 g for 5 min in PBS. (2) The cells after centrifugation were taken, and the supernatant was discarded. 50 ⁇ l of Geltrex was added to the cell pellet, mixed, and used, and Wnt10b factor was added to make the concentration in Geltrex 1 ug/ml.
  • the histomorphological identification of the stem tissue transplanted skin tissue of the experimental group and the control group in Example 3 and Example 4 was as follows: Preparation of epidermal stem cell regeneration hair follicle structure tissue section: (1) Excision Example 3 and Example 4, respectively The skin tissue area of the stem cell transplantation of the middle experimental group and the control group was fixed with 4% paraformaldehyde overnight, immersed in PBS for 3 hours, and dehydrated with 30% sucrose for 8 hours. (2) OCT embedded tissue, frozen sections, and tissue sections of 10 ⁇ m thickness were taken. (3) Dry the tissue section naturally. (4) Tissue sections were washed three times with PBS for 5 minutes each time. (5) Tissue sections were diafiltered for 10 minutes with 0.2-0.5% triton X-100 (in PBS).
  • tissue sections after the membrane treatment were washed three times with PBS for 5 minutes each time.
  • Tissue sections were blocked with 2% BSA for 30 minutes and then washed twice with PBS.
  • the blocked tissue sections were added to the avidin Cy3 labeled 1 antibody and incubated for 3 hours at room temperature.
  • the tissue sections to which the primary antibody was ligated were washed three times with PBS for 5 minutes each time.
  • Dyeing was carried out by adding 0.5 ⁇ g/ml DAPI (in PBS preparation) for 10 minutes.
  • the stained tissue sections were washed three times with PBS to remove excess DAPI.
  • Tissue sections from which excess DAPI was removed were added to a 20 ⁇ l capsule seal.
  • tissue dyeing sample with good sealing is placed in a 4 degree refrigerator and can be stored for more than 2 weeks.
  • Histomorphological identification of tissue sections Tissue sections were observed by confocal microscopy. It was observed that the hair follicle structure was formed in the regenerated skin tissue of Example 2, and its structural morphology was similar to that of normal skin hair follicles; The staining results of the labeled MAB1281 showed that the cells of the regenerated hair follicles all showed MAB1281 positive, indicating that the newborn hair follicles were differentiated from the transplanted human pluripotent stem cells (results refer to Figure 5).

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Abstract

A method for obtaining an artificial hair follicle. The method comprises: transplanting human pluripotent stem cells into the skin of a heterologous embryo at the hair forming stage; continuously cultivating the embryo transplanted with the human pluripotent stem cells in a heterologous maternal body until the embryo is delivered; and isolating hair follicles in the skin of the embryo transplanted with the human pluripotent stem cells to obtain an artificial hair follicle.

Description

人工毛囊及其制备方法和应用Artificial hair follicle and preparation method and application thereof 技术领域Technical field
本发明涉及生物技术领域,具体地,本发明涉及人工毛囊及其制备方法和应用。The present invention relates to the field of biotechnology, and in particular, to artificial hair follicles and methods for their preparation and use.
背景技术Background technique
脱发导致的头发稀少,已经成为困扰现代男性(包括部分女性)越来越严重的亚健康问题,脱发虽然不会严重的生理健康为题,但是脱发会严重的影响个人的心理状态,所以会严重的降低个人的生活质量。据不完全统计,我国目前有超过2亿人存在不同程度的脱发问题,在全球该类人群体已经超过10亿。病理性异常头发脱落,如男性的秃顶,是在某些因素的作用下毛囊长期处于休止期而无法进入生长周期,现在临床上针对病理性脱发的药物甚少,而且这些药物也是只针对很少比例的患者有作用,比如米诺地尔和非那雄胺,都是临床最广泛应用的两种防脱发药物,是两种药物不仅要长期服用,有不同程度的副作用,同时防脱效果也比较微弱。Hair loss caused by hair loss has become a sub-health problem that is plaguing modern males (including some females). Although hair loss does not cause serious physical health problems, hair loss can seriously affect an individual's psychological state, so it will be serious. Reduce the quality of life of individuals. According to incomplete statistics, there are currently more than 200 million people in China who have different degrees of hair loss. In the world, this group of people has exceeded 1 billion. Pathological abnormal hair loss, such as male baldness, is caused by certain factors, the hair follicles are in a long-term rest period and cannot enter the growth cycle. Now there are few clinically targeted drugs for pathological alopecia, and these drugs are only for very few. The proportion of patients with effects, such as minoxidil and finasteride, are the two most widely used anti-hair loss drugs in clinical practice. It is not only for long-term use, but also for different degrees of side effects. Relatively weak.
干细胞(stem cells)是人体及其各种组织细胞的初始来源,其最显著的生物学特征是既有自我更新和不断增殖的能力,又有多向分化的潜能。干细胞根据不同的来源分为成体干细胞(somaticstem cells)和胚胎干细胞(embryonic stem cells,ES细胞)。成体干细胞包括骨髓间充质干细胞、胰腺干细胞、神经干细胞等,在成体组织中存在的。2006年,日本京都大学中山伸弥(Yamanaka)研究小组采用体外基因转染技术,从24个因子中筛选出Oct4、Sox2、c-Myc、Klf4等4个转录因子,通过逆转录病毒将上述4个转录因子导入胚胎小鼠成纤维细胞或成年小鼠尾部皮肤成纤维细胞,在小鼠ES(胚胎干细胞)细胞的培养条件下获得了Fbx15+的多潜能干细胞系,这类iPS细胞在细胞形态、增殖能力、表面抗原标志、基因表达谱、多潜能干细胞特异性基因的表观遗传学状态、端粒酶活性等方面与hES细胞相似,并且在体外培养时和在小鼠体内畸胎瘤形成中均可分化为3个胚层的不同细胞类型(Takahashi K et al.Cell 2007;131:861-872)。这一被学界称为生物科学“里程碑”的重大突破有望帮助科学家绕过克隆技术的伦理、道德纷争,为医学应用打开大门。Stem cells are the initial source of the human body and its various tissue cells. Its most prominent biological characteristics are its ability to self-renew and proliferate, as well as the potential for multi-directional differentiation. Stem cells are classified into adult stem cells (emmatic stem cells) and embryonic stem cells (ES cells) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, and the like, which are present in adult tissues. In 2006, the Yamanaka team at Kyoto University in Japan used in vitro gene transfection technology to screen four transcription factors including Oct4, Sox2, c-Myc, and Klf4 from 24 factors, and the above four by retrovirus. The transcription factor is introduced into embryonic mouse fibroblasts or adult mouse tail skin fibroblasts, and Fbx15+ pluripotent stem cell lines are obtained under the condition of mouse ES (embryonic stem cell) cells. The cell morphology and proliferation of such iPS cells are obtained. The ability, surface antigen marker, gene expression profile, epigenetic state of pluripotent stem cell-specific genes, and telomerase activity are similar to those of hES cells, and both in vitro culture and in teratoma formation in mice. Different cell types can be differentiated into three germ layers (Takahashi K et al. Cell 2007; 131: 861-872). This major breakthrough, known as the “milestone” of the biological sciences, is expected to help scientists bypass the ethical and moral disputes of cloning technology and open the door to medical applications.
自20世纪90年代中期以来,椭圆形头皮切割采集是获得毛发移植毛囊集落的首选方法。在过去20年中,毛囊单位提取(FUE)已成为越来越受欢迎的获得供者头发的方法(Rassman,Bernstein et al.2002,Facial Plast Surg Clin North Am,Harris 2013,Dermatol Surg.)。FUE使用手动或机器人装置来从供体区域获取独立毛囊单位(Cole2013,Facial Plast Surg Clin North Am.),FUE相对于椭圆供体收获的主要优点是无供体头发收获然后缝合或缝合封闭的线性瘢痕。这是一个很大的优势,特别是对于头发短的执法者,也不会看到很明显的伤痕。虽然FUE具有无线性瘢痕的优点,仍然存在其他方面的问题和潜在的长期副作用。与全层皮肤切口一样,收获每个毛囊单位会产生一 个1mm直径的冲头的瘢痕。这些瘢痕会对剩余毛囊的着色及发育有影响。此外,无论FUE是通过手动,机动或机器人打孔进行的,与供体椭圆头皮切割收获毛囊一样,存在来自供体区域的头发明显消耗的风险,即供体区域的毛发会明细减少。这可能会产生医源性“蛾食”或“伪梅毒”外观(
Figure PCTCN2018074827-appb-000001
N et al.J Plast Reconstr Aesthet Surg.2012,Poswal A et al.Indian J Dermatol.2011)。FUE是获得供者头发的有效和有用的方式。它产生比椭圆形头皮切割供体毛囊获取会产生少的可见瘢痕。与任何手术技术一样,该方法存在局限性和副作用。
Since the mid-1990s, oval scalp cutting has been the preferred method of obtaining hair follicle colonies. In the past 20 years, follicular unit extraction (FUE) has become an increasingly popular method of obtaining donor hair (Rassman, Bernstein et al. 2002, Facial Plast Surg Clin North Am, Harris 2013, Dermatol Surg.). FUE uses manual or robotic devices to obtain independent follicular units from the donor area (Cole 2013, Facial Plast Surg Clin North Am.). The main advantage of FUE relative to elliptical donor harvesting is that there is no donor hair harvesting and then stitching or stitching closed linearity. scar. This is a big advantage, especially for law enforcement short hair, and will not see obvious scars. Although FUE has the advantage of wireless scarring, there are still other problems and potential long-term side effects. As with full-thickness skin incisions, harvesting each follicular unit produces a scar of a 1 mm diameter punch. These scars have an effect on the coloration and development of the remaining hair follicles. Furthermore, whether the FUE is performed by manual, motorized or robotic perforation, as with the donor elliptical scalp cutting harvesting hair follicles, there is a risk of significant consumption of hair from the donor area, ie the hair of the donor area will be reduced in detail. This may result in an iatrogenic "moth" or "pseudo-syphilis" appearance (
Figure PCTCN2018074827-appb-000001
N et al. J Plast Reconstr Aesthet Surg. 2012, Poswal A et al. Indian J Dermatol. 2011). FUE is an effective and useful way to get the donor's hair. It produces less visible scars than the elliptical scalp cut donor hair follicles. As with any surgical technique, this approach has limitations and side effects.
总之,除了药物治疗,毛囊移植成为近年来新兴的解决脱发问题的方案。但是毛囊移植的方法是取后枕部的毛囊后,再将该毛囊移植到同一个人的前额头发稀少的位置,虽然该方法立竿见影,见效很快,但是毕竟是如同拆东墙补西墙,只对少量脱发的患者有效,但对于大面积脱发的患者也不能有好的治疗效果。同时毛囊提取是会对毛囊产生机械性损伤,影响毛囊的存活率。In short, in addition to medical treatment, hair follicle transplantation has become an emerging solution to the problem of hair loss in recent years. However, the method of hair follicle transplantation is to take the hair follicle of the posterior occipital region and then transplant the hair follicle to the same person's forehead hair. The method is immediate and effective, but after all, it is like disassembling the east wall to make up the western wall. It is effective for patients with a small amount of hair loss, but it cannot have a good therapeutic effect for patients with large area hair loss. At the same time, hair follicle extraction is a mechanical damage to the hair follicle, affecting the survival rate of the hair follicle.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明提出了一种基于人自体干细胞和异种载体的毛囊再生方法。The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the present invention proposes a hair follicle regeneration method based on human autologous stem cells and heterogeneous vectors.
在本发明的第一方面,本发明提出了一种获得人工毛囊的方法。根据本发明的实施例,所述方法包括:将人多能干细胞移植入异种胚胎的皮肤中,所述异种胚胎处于毛发形成阶段;将移植有人多能干细胞的胚胎在异种母体内继续进行培养,直至胚胎娩出;以及分离胚胎中移植有人多能干细胞的皮肤中的毛囊,以便获得所述人工毛囊。根据本发明实施例的方法获得的人工毛囊数量多,结构形态与正常皮肤中的毛囊类似,可用于人不同部位的毛囊移植,如头皮、眉毛、胡须、体毛等;同时获得的人工毛囊移植回到多能干细胞提供者不会出现免疫排斥反应,安全性大大提高。In a first aspect of the invention, the invention proposes a method of obtaining artificial hair follicles. According to an embodiment of the present invention, the method comprises: transplanting human pluripotent stem cells into the skin of a heterologous embryo, the heterologous embryos being in a hair formation stage; and continuing to culture the embryos transplanted with the pluripotent stem cells in the xenogeneic mother, Until the embryo is delivered; and the hair follicle in the skin of the pluripotent stem cells is transplanted in the embryo to obtain the artificial hair follicle. The artificial hair follicle obtained by the method according to the embodiment of the invention has a large number, and the structural morphology is similar to that of the hair follicle in normal skin, and can be used for hair follicle transplantation of different parts of the human body, such as scalp, eyebrow, beard, body hair, etc.; Until the pluripotent stem cell provider does not have immune rejection, the safety is greatly improved.
根据本发明的实施例,上述方法还可以进一步包括如下附加技术特征至少之一:According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例,所述人多能干细胞为诱导多能干细胞(iPSCs),所述移植之前进一步包括:将所述诱导多能干细胞分化为皮肤类器官,所述皮肤类器官包括外胚层细胞,中胚层细胞和神经内胚层细胞。干细胞要首先分化为3个胚层,再进一步分化为皮肤类器官,从而进一步分化为毛囊结构。According to an embodiment of the present invention, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs), and the transplanting further comprises: differentiating the induced pluripotent stem cells into skin-like organs, the skin-like organs including ectoderm Cells, mesoderm cells and neuroendodermal cells. The stem cells first differentiate into three germ layers, and then further differentiate into skin-like organs, thereby further differentiating into hair follicle structures.
根据本发明的实施例,将所述诱导多能干细胞分化为皮肤类器官包括:将所述诱导多能干细胞进行悬浮培养,以便得到3D细胞球;将所述3D细胞球进一步进行分化培养,以便获得皮肤类器官。According to an embodiment of the present invention, the differentiation of the induced pluripotent stem cells into a dermatophyte comprises: suspending the induced pluripotent stem cells to obtain a 3D cell sphere; and further culturing the 3D cell sphere so that Get skin-like organs.
所述诱导多能干细胞来源不受特别限制,可以源于任意一种成体细胞。根据本发明的实施例,所述诱导多能干细胞来源于血液细胞或皮肤细胞。The source of the induced pluripotent stem cells is not particularly limited and may be derived from any one of adult cells. According to an embodiment of the invention, the induced pluripotent stem cells are derived from blood cells or skin cells.
根据本发明的实施例,将所述诱导多能干细胞分化为皮肤类器官是在所述多能干细胞与 BMP-4,SB431542,FGF-2和LND接触的条件下进行的。According to an embodiment of the present invention, the differentiation of the induced pluripotent stem cells into a dermatophyte is carried out under conditions in which the pluripotent stem cells are contacted with BMP-4, SB431542, FGF-2 and LND.
根据本发明的实施例,所述BMP-4的浓度为50ng/mL。进而进一步提高了皮肤类器官的诱导效率。According to an embodiment of the invention, the concentration of BMP-4 is 50 ng/mL. Further, the induction efficiency of the skin-like organs is further improved.
根据本发明的实施例,所述移植之前进一步包括:将所述皮肤类器官进行消化处理,以便获得单细胞混合液。According to an embodiment of the present invention, the transplanting further comprises: digesting the skin-like organ to obtain a single-cell mixture.
根据本发明的实施例,将所述单细胞混合液与基质胶或凝胶类物质混合。进而所述单细胞混合液可在促进毛囊形成的因子的诱导下形成膜状细胞层。根据本发明的实施例,所述膜状细胞层的厚度与受体异种胚胎的皮肤厚度相同。According to an embodiment of the invention, the single cell mixture is mixed with a matrigel or gel-like substance. Further, the single cell mixture can form a membranous cell layer under the induction of a factor that promotes hair follicle formation. According to an embodiment of the invention, the thickness of the membranous cell layer is the same as the thickness of the skin of the recipient xenogenic embryo.
根据本发明的实施例,所述基质胶包括选自Geltrex和ColI的至少之一。According to an embodiment of the invention, the Matrigel comprises at least one selected from the group consisting of Geltrex and ColI.
根据本发明的实施例,所述人多能干细胞包括选自毛囊干细胞、皮肤干细胞及其它具有毛囊形成或诱导能力的干细胞。According to an embodiment of the invention, the human pluripotent stem cells comprise stem cells selected from the group consisting of hair follicle stem cells, skin stem cells, and other hair follicle forming or inducing abilities.
根据本发明的实施例,所述人多能干细胞包括基因修饰的多能干细胞或诱导多能干细胞。任选地,所述基因修饰为基因敲除或基因表达。According to an embodiment of the invention, the human pluripotent stem cells comprise genetically modified pluripotent stem cells or induced pluripotent stem cells. Optionally, the genetic modification is gene knockout or gene expression.
根据本发明的实施例,所述移植之前进一步包括:将所述人多能干细胞与促进毛囊形成的因子混合。进而毛囊形成效率大大提高。According to an embodiment of the invention, the transplanting further comprises: mixing the human pluripotent stem cells with a factor that promotes hair follicle formation. Furthermore, the hair follicle formation efficiency is greatly improved.
根据本发明的实施例,所述促进毛囊形成的因子为Wnt10b因子。进而毛囊形成效率进一步提高。According to an embodiment of the invention, the factor that promotes hair follicle formation is a Wnt10b factor. Furthermore, the hair follicle formation efficiency is further improved.
根据本发明的实施例,所述Wnt10b因子的浓度为1ug/mL。进而毛囊形成效率进一步提高。According to an embodiment of the invention, the concentration of the Wnt10b factor is 1 ug/mL. Furthermore, the hair follicle formation efficiency is further improved.
根据本发明的实施例,所述异种胚胎包括选自猪胚胎、灵长类动物胚胎、兔胚胎、牛胚胎以及羊胚胎的至少之一。需要说明的是,所述异种动物载体在使用时必须达到临床卫生标准,既不能携带任何临床明确指定的具有致病风险的病原体。需要进一步说明的是,所述异种胚胎只是为人体干细胞发育成毛囊提供环境,最后分离发育成熟的人工毛囊时需要完全去除所有动物源性的组织和细胞。According to an embodiment of the invention, the xenogeneic embryo comprises at least one selected from the group consisting of a pig embryo, a primate embryo, a rabbit embryo, a bovine embryo, and a sheep embryo. It should be noted that the heterologous animal carrier must meet clinical hygiene standards when used, and can neither carry any clinically specified pathogens at risk of disease. It should be further noted that the heterogeneous embryo only provides an environment for the development of human stem cells into hair follicles, and finally it is necessary to completely remove all animal-derived tissues and cells when separating mature artificial hair follicles.
根据本发明的实施例,所述分离是在胚胎分娩后的第0~6个月内进行的。此时的毛囊具有较为完整的结构,具备移植条件且再生能力强。According to an embodiment of the invention, the separation is carried out within 0 to 6 months after the embryo is delivered. The hair follicle at this time has a relatively complete structure, has a transplant condition and is highly regenerative.
根据本发明的实施例,进一步包括将所获得人工毛囊移植回到干细胞提供者皮肤后,判断该毛囊移植后是否能够存活且有正常的生长周期。如果毛囊处于一个生长期3个月以上的,同时进入休止期后会在1-3个月内进入生长期,即判断该毛囊移植后存活且有正常的生长周期。即可用于后续的毛囊移植。According to an embodiment of the present invention, further comprising, after transplanting the obtained artificial hair follicle back to the skin of the stem cell provider, determining whether the hair follicle is viable after transplantation and has a normal growth cycle. If the hair follicle is in a growth period of more than 3 months, and enters the growth period within 1-3 months after entering the rest period, it is judged that the hair follicle survives after transplantation and has a normal growth cycle. It can be used for subsequent hair follicle transplantation.
在本发明的第二方面,本发明提出了一种人工毛囊。根据本发明的实施例,所述毛囊是由前面所述的方法获得的。根据本发明实施例的人工毛囊可以从整个皮肤组织中剥离出来, 可以最大程度减小毛囊的损伤;同时根据本发明实施例的人工毛囊是自体干细胞产生的自体毛囊,如果将这些毛囊移植回到干细胞提供者,不会出现免疫排斥反应,安全性更高。In a second aspect of the invention, the invention proposes an artificial hair follicle. According to an embodiment of the invention, the hair follicle is obtained by the method described above. The artificial hair follicle according to the embodiment of the present invention can be peeled off from the entire skin tissue, and the damage of the hair follicle can be minimized; and the artificial hair follicle according to the embodiment of the present invention is an autologous hair follicle produced by the autologous stem cell, if these hair follicles are transplanted back Stem cell providers do not have immune rejection and are safer.
在本发明的第三方面,本发明提出了一种毛囊种植的方法。根据本发明的实施例,所述方法包括将前面所述的人工毛囊接种到自体皮肤表面。根据本发明实施例的方法可应用于男性脱发(谢顶)的毛囊种植、头发加密使用的毛囊种植、发际线调整使用的毛囊种植、疤痕性头皮的毛囊种植,也包括眉毛种植,胡须种植和体毛种植。In a third aspect of the invention, the invention proposes a method of hair follicle planting. According to an embodiment of the invention, the method comprises inoculating the artificial hair follicles described above onto the surface of the autologous skin. The method according to an embodiment of the present invention can be applied to hair follicle planting of male hair loss (sharp top), hair follicle planting for hair encryption, hair follicle planting for hairline adjustment, hair follicle planting of scar scalp, eyebrow planting, and beard planting. And body hair planting.
需要说明的是,本发明所述的“移植”、“接种”操作,不受特别限制,本领域技术人员即可轻松完成,不需专业培训。It should be noted that the “transplantation” and “inoculation” operations described in the present invention are not particularly limited, and can be easily accomplished by those skilled in the art without professional training.
附图说明DRAWINGS
图1是根据本发明实施例的基于自体诱导多能干细胞和异种载体的毛囊再生方法流程图;1 is a flow chart of a hair follicle regeneration method based on autologous induced pluripotent stem cells and xenogeneic vectors according to an embodiment of the present invention;
图2是根据本发明实施例的iPS细胞向皮肤细胞的诱导分化图,2 is a diagram showing induced differentiation of iPS cells into skin cells according to an embodiment of the present invention,
其中,a,从血液中分离得到了单个核细胞;b,把单个核细胞诱导成为iPS细胞,iPS细胞形成克隆;c,iPS细胞向皮肤前体细胞分化;Wherein, a, mononuclear cells are isolated from the blood; b, mononuclear cells are induced into iPS cells, iPS cells form clones; c, iPS cells differentiate into skin precursor cells;
图3是根据本发明实施例的分化的iPS细胞与凝胶混合,及细胞的移植;3 is a mixture of differentiated iPS cells and a gel, and transplantation of cells according to an embodiment of the present invention;
图4是根据本发明实施例的移植后的干细胞形成毛囊结构的结果图;以及4 is a diagram showing the results of formation of a hair follicle structure by stem cells after transplantation according to an embodiment of the present invention;
图5是根据本发明实施例的人源细胞特异性标记MAB1281染色显示形成的毛囊和皮肤结构全来自人源干细胞的结果图。Figure 5 is a graph showing the results of staining of hair follicles and skin structures formed entirely from human stem cells by human cell-specific marker MAB1281 staining in accordance with an embodiment of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.
本发明提供了一种基于人自体干细胞和异种载体的毛囊再生方法,其中,毛囊再生的方法包括人体细胞或干细胞的分离、培养和诱导,其中得到干细胞的方法有两种,第一种是直接分离脱发患者的毛囊干细胞或皮肤细胞;第二种是将成体细胞(如血细胞和皮肤细胞)诱导成为具有多向分化潜力的多功能干细胞,该技术即为诱导性多能干细胞(iPS细胞),即获得脱发患者或需要头发移植者的诱导多能干细胞,该技术目前已经有有效的实现手段。获得诱导多能干细胞后首先将其分化为外胚层细胞,再与基质胶或凝胶类物质混合,同时加入能促进外胚层细胞向毛囊分化和发育的因子,让诱导后的细胞和凝胶类支架物质形成膜状细胞层。该膜状细胞层的厚度应该和受体异种动物胎儿皮肤厚度相同。此时由于受体动物胎儿皮肤中的毛囊正处于生成和发育阶段,所以其微环境最适合毛囊再生,所以移植后的已经分化为外胚层的人体干细胞再毛囊分化诱导因子的作用下会有效的形成毛囊。对 于毛囊干细胞,在移植前则不需要向外胚层分化的诱导,但为了促进毛囊形成的效率、仍需要在移植前与促进毛囊形成的因子混合。The invention provides a hair follicle regeneration method based on human autologous stem cells and a heterogeneous vector, wherein the method for regenerating hair follicles comprises the separation, culture and induction of human cells or stem cells, wherein there are two methods for obtaining stem cells, the first one is direct Separating hair follicle stem cells or skin cells from alopecia patients; the second is to induce adult cells (such as blood cells and skin cells) into pluripotent stem cells with multipotential differentiation potential, which is induced pluripotent stem cells (iPS cells). That is, to obtain a hair loss patient or an induced pluripotent stem cell requiring a hair transplanter, the technique has been effectively implemented. After the induced pluripotent stem cells are obtained, they are first differentiated into ectodermal cells, mixed with Matrigel or gel-like substances, and factors that promote the differentiation and development of ectodermal cells to hair follicles are added, and the induced cells and gels are added. The scaffold material forms a membranous cell layer. The thickness of the membranous cell layer should be the same as the thickness of the fetal skin of the recipient xenobiotic. At this time, since the hair follicle in the fetal skin of the recipient animal is in the stage of formation and development, the microenvironment is most suitable for hair follicle regeneration, so the human stem cell which has been differentiated into the ectoderm after transplantation will be effective under the action of hair follicle differentiation inducing factor. Form hair follicles. For hair follicle stem cells, induction of ectodermal differentiation is not required prior to transplantation, but in order to promote the efficiency of hair follicle formation, it is still necessary to mix with factors that promote hair follicle formation before transplantation.
载体动物胎儿出生时,其皮肤中移植的人体干细胞已经部分发育成了毛囊,此时的毛囊已经具有较为完整的结构,部分毛囊已经有毛发长出。此时的毛囊已经具备移植条件,所以可以在载体动物胎儿出生时就分离人体毛囊,然后种植。也可以在载体动物胎儿出生饲养一段时间,让其携带的人体毛囊进一步的发育成熟,然后再分离种植。异种载体动物包括不同品种猪,灵长类动物及兔,牛,羊。但是考虑到伦理和研究背景及安全性,达到医疗级卫生水平的猪应该是优先使用的动物。所有使用异种动物载体在实际应用时都必须达到临床卫生标准,既不能携带任何临床明确指定的具有致病风险的病原体。对于猪来说,要使用不携带内源性逆转录病毒的品系。When the carrier animal is born, the human stem cells transplanted in the skin have partially developed into hair follicles. At this time, the hair follicles have a relatively complete structure, and some hair follicles have hair growth. At this time, the hair follicles already have the transplantation conditions, so the human hair follicles can be separated when the carrier animal fetus is born, and then planted. The carrier animal fetus can also be raised for a period of time, allowing the human hair follicles carried by it to further mature and then planted separately. Heterogeneous carrier animals include different breeds of pigs, primates and rabbits, cattle, and sheep. However, given the ethical and research background and safety, pigs that achieve medical-grade hygiene should be the preferred animals. All use of heterologous animal carriers must meet clinical hygiene standards in practice, and should not carry any clinically specified pathogens at risk of disease. For pigs, strains that do not carry endogenous retroviruses are used.
通过该发明的人工毛囊有诸多实际应用方向,其中包括用于治疗男性脱发(谢顶)的毛囊种植,用于头发加密使用的毛囊种植,用于发际线调整使用的毛囊种植,用于疤痕性头皮的毛囊种植,也包括眉毛种植,胡须种植和体毛种植。由于该发明的特点是通过自体干细胞再生的全新毛囊,所以其在应用过程中具有一下几方面的优势,首先,通过培养后的后的干细胞或诱导性多功能干细胞可以在数量上大量扩增,所以理论上该方法可以获得任意数量的毛囊;干细胞在载体动物中发育阶段可以加入不同的生长因子,控制毛囊生长为不同大小和形状,从而用于不同部位的毛囊再生,如眉毛和胡须等;本发明中分离毛囊会从整个皮肤组织中剥离毛囊,从而可以最大程度减小对毛囊的损伤;本发明再生的毛囊是自体干细胞产生的自体毛囊,所以在将这些毛囊移植回到干细胞提供者不会出现免疫排斥反应。The artificial hair follicles of the invention have many practical application directions, including hair follicle cultivation for treating male hair loss (Xieding), hair follicle cultivation for hair encryption, hair follicle cultivation for hairline adjustment, and scarring. Hair follicle cultivation of the scalp, including eyebrow planting, beard planting and body hair planting. Since the invention is characterized by a new hair follicle regenerated by autologous stem cells, it has several advantages in application. First, the post-cultured stem cells or induced pluripotent stem cells can be quantitatively expanded in a large amount. Therefore, in theory, the method can obtain any number of hair follicles; stem cells can be added with different growth factors in the development stage of the carrier animal, and the hair follicles can be controlled to grow into different sizes and shapes, thereby being used for hair follicle regeneration in different parts, such as eyebrows and beards; In the present invention, the separation of the hair follicle peels the hair follicle from the entire skin tissue, thereby minimizing the damage to the hair follicle; the regenerated hair follicle of the present invention is an autologous hair follicle produced by the autologous stem cell, so the transplantation of the hair follicle back to the stem cell provider is not An immune rejection will occur.
为了便于理解,根据本发明实施例的基于自体诱导多能干细胞和异种载体的毛囊再生方法流程图参考图1。For ease of understanding, a flow chart of a hair follicle regeneration method based on autologous induced pluripotent stem cells and xenogeneic vectors according to an embodiment of the present invention is referred to FIG.
下面详细描述本发明的具体实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。Specific embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.
实施例1Example 1
1、细胞提供者的外周血细胞准备,诱导和检测:1. Preparation, induction and detection of peripheral blood cells of the cell provider:
从细胞供体者中通过静脉抽血分离出30ml外周血,通过Ficoll-Hypaque密度梯度分离获得单核(MNC)细胞。将MNC细胞在含有10%FBS和10ng/ml IL-7或10ng/ml G-CSF,GM-CSF,IL-3和IL-6的α-MEM培养基中培养5天。随后这些细胞用于产生诱导性多能干细胞,具体步骤如下,将1-2×10 4个MNC置于96孔板的1个孔中,并用CytoTune-iPS重编程试剂盒(DNAVEC公司,Tsukuba,日本)感染编码OCT4,SOX2,KLF4,cMYC F-缺 陷型仙台病毒载体(Sev/ΔF),其中每个因素的感染倍数(MOI)为5-10。感染后一天,低速离心去掉培养液,收获细胞,并将其培育在用小鼠胚胎成纤维细胞(MEF)铺底培养的六孔板中,其所用培养基中与上述培养基相同,培养另外一天。第2天,将培养基更换为补充有8ng/ml bFGF的人ES(人胚胎干细胞)培养基,并每天用新鲜培养基换液。形态学类似于ES克隆在感染后第13天左右开始出现;在第21或28天挑取克隆,扩大培养,并通过免疫荧光或流式细胞分析检查多能性标记。其中免疫荧光的检测多能性标记的方法为,将iPS细胞用D-PBS洗涤3次并在室温下在4%多聚甲醛中固定10分钟。用D-PBS洗涤后,用甲醇使细胞破膜(室温1分钟),D-PBS洗涤并在PBS-5%驴血清中封闭30分钟。将抗Oct4(小鼠单克隆),Tra1-81(小鼠单克隆)和Nanog的抗体在4℃温育孵育细胞过夜。用D-PBS将细胞洗涤3次,并在室温下进行合适的荧光二抗(Invitrogen)1小时,然后DAPI染色1分钟。在荧光显微镜下检测多能性标记表达情况。畸胎瘤实验的方法为,用胶原酶处理(1mg/ml)收集血液来源的iPS细胞,并通过细胞聚集沉降将iPS细胞从滋养层细胞中分离。再用D-PBS洗涤细胞,重悬于500μl人ES细胞培养基中并皮下注射到SCID小鼠(Taconic)中。注射4-6周后,将肿瘤从安乐死的小鼠中取出,并在4%多聚甲醛中固定。根据苏木精和伊红染色,将样品石蜡包埋,切片分析。 30 ml of peripheral blood was isolated from the cell donor by venous blood, and mononuclear (MNC) cells were obtained by Ficoll-Hypaque density gradient separation. MNC cells were cultured for 5 days in α-MEM medium containing 10% FBS and 10 ng/ml IL-7 or 10 ng/ml G-CSF, GM-CSF, IL-3 and IL-6. These cells are then used to produce induced pluripotent stem cells by following the steps below, placing 1-2×10 4 MNCs in one well of a 96-well plate and using the CytoTune-iPS reprogramming kit (DNAVEC, Tsukuba, Japan) Infected with OCT4, SOX2, KLF4, cMYC F-deficient Sendai virus vector (Sev/ΔF), each of which has a multiplicity of infection (MOI) of 5-10. One day after the infection, the culture solution was removed by low-speed centrifugation, and the cells were harvested and cultured in a six-well plate cultured with mouse embryonic fibroblasts (MEF) in the same medium as the above medium, and cultured for another day. . On day 2, the medium was changed to human ES (human embryonic stem cell) medium supplemented with 8 ng/ml bFGF, and changed daily with fresh medium. Morphology similar to ES cloning began to appear around day 13 post infection; clones were picked on day 21 or 28, expanded, and pluripotency markers were examined by immunofluorescence or flow cytometry. The method for detecting pluripotency markers by immunofluorescence was to wash iPS cells three times with D-PBS and fix them in 4% paraformaldehyde for 10 minutes at room temperature. After washing with D-PBS, the cells were ruptured with methanol (room temperature for 1 minute), washed with D-PBS and blocked in PBS-5% sputum serum for 30 minutes. Anti-Oct4 (mouse monoclonal), Tra1-81 (mouse monoclonal) and Nanog antibodies were incubated overnight at 4 °C. The cells were washed 3 times with D-PBS and subjected to a suitable fluorescent secondary antibody (Invitrogen) for 1 hour at room temperature, followed by DAPI staining for 1 minute. The expression of pluripotency markers was examined under a fluorescence microscope. The teratoma experiment was performed by collecting collagen-derived (1 mg/ml) blood-derived iPS cells and separating iPS cells from trophoblast cells by cell aggregation sedimentation. The cells were washed again with D-PBS, resuspended in 500 μl of human ES cell culture medium and injected subcutaneously into SCID mice (Taconic). After 4-6 weeks of injection, tumors were removed from euthanized mice and fixed in 4% paraformaldehyde. The samples were embedded in paraffin and sectioned for analysis according to hematoxylin and eosin staining.
2、iPS诱导多能细胞体外分化的方法2. Method for inducing pluripotent cell differentiation in vitro by iPS
用1mg/ml胶原酶IV(Invitrogen)处理iPSC克隆10分钟,用D-PBS洗涤,通过刮擦和移液几次将其解离成团块。将iPSC块在不含bFGF的ES培养基中涂布到Lipidure涂层板(NOF Corporation,Irvine,CA,美国)。每天更换培养基一次。在悬浮培养8天后,将胚状体(EB)转移到明胶包被的平板中,并在相同的培养基中再培养8-10天。然后分析细胞标记的表达情况。The iPSC clones were treated with 1 mg/ml collagenase IV (Invitrogen) for 10 minutes, washed with D-PBS, and dissociated into agglomerates by scraping and pipetting several times. The iPSC blocks were coated onto Lipidure coated plates (NOF Corporation, Irvine, CA, USA) in ES medium without bFGF. Change the medium once a day. After 8 days of suspension culture, embryoid bodies (EB) were transferred to gelatin-coated plates and cultured for an additional 8-10 days in the same medium. The expression of the cell marker is then analyzed.
3、诱导多能细胞的核型分析过程3. The karyotype analysis process of induced pluripotent cells
在将人iPSC传代培养后的一天,将细胞暴露于0.25μg/ml的凝胶中3.5小时,消化,收集并暴露于低渗溶液(0.4%柠檬酸钠:0.4%KCl=1:1)中16分钟。将细胞用甲醇/乙酸(3:1)固定2次,共1小时。然后将细胞滴在冷湿和干净的玻璃片上。将载玻片在70℃下孵育4小时。然后将载玻片用0.01%胰蛋白酶在37℃下处理10-12秒,用0.9%NaCl洗涤,然后在37℃用Giemsa溶液(Giemsa:磷酸盐缓冲液=1.5ml:40ml,pH7.4)染色,2.5分钟。通过显微镜检查确定核型。One day after subculture of human iPSCs, cells were exposed to 0.25 μg/ml gel for 3.5 hours, digested, collected and exposed to hypotonic solution (0.4% sodium citrate: 0.4% KCl = 1:1). 16 minutes. The cells were fixed twice with methanol/acetic acid (3:1) for 1 hour. The cells were then dropped onto cold, wet and clean glass slides. Slides were incubated for 4 hours at 70 °C. The slides were then treated with 0.01% trypsin at 37 ° C for 10-12 seconds, washed with 0.9% NaCl, then with Giemsa solution at 37 ° C (Giemsa: phosphate buffer = 1.5 ml: 40 ml, pH 7.4) Dyeing, 2.5 minutes. The karyotype was determined by microscopic examination.
实施例2Example 2
1、iPS细胞向皮肤类器官的分化无滋养层培养的准备1. Preparation of iPS cells to skin-like organs for trophoblast-free culture
该操作需要在使用无菌的生物安全柜中进行。与Matrigel类似,Geltrex基质在室温(RT) 下快速凝固,因此需要在将新的胶进行分装,并在分装时预先冷冻的吸头,支架和管。制作50,100和200μL等分分装,并将其储存在-80℃。以1:100稀释使用Geltrex。无饲养层iPSC培养只需要Geltrex作为表面包被剂,然而对于iPSC分化,Geltrex和ColI的组合对于诱导iPS细胞向皮肤类器官的分化更有效。对于60mm组织培养皿Geltrex表面包被方法如下。如果要使用较大的培养皿,相应地调节Geltrex溶液的体积。从-80℃冰箱中取出50μLGeltrex,并将其置于生物安全柜内的冰上。加入5mL冷却无菌DMEM/F12至15mL离心管中。使用1mL玻璃移液管,从15mL离心管中取1mL冷DMEM/F12,并加入冷冻的Geltrex。轻轻上下吸管完全溶解Geltrex,然后将溶解的Geltrex转移到离心管中的DMEM/F12的其余部分,混合稀释的Geltrex。将50μL 3mg/mL ColI储备溶液加入到稀释的Geltrex中。将稀释的Geletrex与ColI混合。加入4毫升上述溶液到60mm的培养皿中。以确保整个表面被涂覆。将培养皿与Geltrex/ColI包被溶液在37℃组织培养箱中孵育至少1小时。涂层完成后,将涂层溶液留在培养皿中,并进行iPSCs的铺板。或者,吸取涂层溶液,并将2mL新鲜的DMEM/F12加入到包被的培养皿中。This operation needs to be performed in a sterile biosafety cabinet. Similar to Matrigel, the Geltrex matrix rapidly solidifies at room temperature (RT), requiring tips, scaffolds and tubes that are dispensed with new gels and pre-frozen at the time of dispensing. Aliquots of 50, 100 and 200 μL were made and stored at -80 °C. Use Geltrex at 1:100 dilution. Graft-free iPSC culture requires only Geltrex as a surface coating agent, whereas for iPSC differentiation, the combination of Geltrex and ColI is more effective in inducing differentiation of iPS cells into skin-like organs. The method of coating the surface of the 60 mm tissue culture dish Geltrex is as follows. If a larger Petri dish is to be used, adjust the volume of the Geltrex solution accordingly. Remove the 50μ LGeltrex from the -80 °C freezer and place it on ice in the biosafety cabinet. Add 5 mL of cooled sterile DMEM/F12 to a 15 mL centrifuge tube. Using a 1 mL glass pipette, 1 mL of cold DMEM/F12 was taken from a 15 mL centrifuge tube and frozen Geltrex was added. Gently dissolve the Geltrex by pipette up and down, then transfer the dissolved Geltrex to the rest of the DMEM/F12 in the centrifuge tube and mix the diluted Geltrex. 50 μL of 3 mg/mL ColI stock solution was added to the diluted Geltrex. The diluted Gelretrex was mixed with ColI. Add 4 ml of the above solution to a 60 mm Petri dish. To ensure that the entire surface is coated. The culture dishes were incubated with the Geltrex/ColI coating solution in a 37 ° C tissue culture incubator for at least 1 hour. After the coating was completed, the coating solution was left in the Petri dish and plated with iPSCs. Alternatively, the coating solution was aspirated and 2 mL of fresh DMEM/F12 was added to the coated Petri dish.
2、iPS细胞的诱导前准备。2. Preparation before induction of iPS cells.
准备一个上述包被处理60mm无滋养层的iPSCs组织培养皿,培养至约70%的融合。在显微镜下检查细胞以确认不存在污染和维持其未分化状态。如果细胞受到外界刺激,它们开始分化,则此类细胞不应用于分化为角化细胞。用于iPSC分化为皮肤类器官,发明人将iPSCs的1:8传代。在37℃水浴中使用N2B27培养基和Dispase预热。使用显微镜,确认克隆数量。从培养皿中轻轻吸出培养基。加入2mL 1xPBS,旋转平板以洗涤细胞,轻轻吸出PBS。加入1mL Dispase,并将培养板放置到37℃组织培养箱中3-5分钟。将上述在使用Geltrex/ColI涂层过程的培养皿中吸出Geltrex/ColI涂层溶液(或DMEM/F12),并将4mL完整的N2B27培养基加入包被的培养皿中。iPS细胞用Dispase孵育3-5分钟后,显微镜确认细胞克隆周围是否有卷曲或折叠。如果消化到位,则将培养板转移到生物安全柜,并小心吸出Dispase。用Dispase处理后,细胞克隆非常松散地附着在培养皿的表面,如果使用太多的力,可能会剥落。轻轻加入2mL纯DMEM/F12。重复洗涤3次。将2mLN2B27完全培养基加入到培养皿中,轻轻地从培养板上刮下菌落。将细胞从培养皿中转移到15mL离心管中,加入6mL N2B27完全培养基,使细胞悬浮液的总体积达到8mL。轻轻混合细胞悬浮液以破碎大块细胞。将1mL细胞悬浮液转移到制备好的的包被培养皿中。将新铺板的细胞转移到培养箱中,轻轻地来回摇晃板,并将其均匀分布。在37℃组织培养箱中孵育细胞过夜。Prepare one of the above-mentioned coated 60 mm trophoblast-free iPSCs tissue culture dishes and incubate to approximately 70% confluence. Cells were examined under a microscope to confirm the absence of contamination and to maintain their undifferentiated state. If cells are stimulated by the outside world and they begin to differentiate, such cells should not be used to differentiate into keratinocytes. For iPSC differentiation into skin-like organs, the inventors passaged 1:8 of iPSCs. Preheat with N2B27 medium and Dispase in a 37 ° C water bath. The number of clones was confirmed using a microscope. The medium was gently aspirated from the Petri dish. 2 mL of 1 x PBS was added, the plate was rotated to wash the cells, and the PBS was gently aspirated. 1 mL of Dispase was added and the plates were placed in a 37 ° C tissue culture incubator for 3-5 minutes. The Geltrex/ColI coating solution (or DMEM/F12) was aspirated above in a Petri dish using the Geltrex/ColI coating process, and 4 mL of intact N2B27 medium was added to the coated Petri dish. After incubating the iPS cells with Dispase for 3-5 minutes, the microscope confirmed whether there was curling or folding around the cell clones. If digested, transfer the plate to the biosafety cabinet and carefully aspirate the Dispase. After treatment with Dispase, the cell clones are very loosely attached to the surface of the culture dish and may peel off if too much force is used. Gently add 2 mL of pure DMEM/F12. Repeat the washing 3 times. 2 mL of N2B27 complete medium was added to the Petri dish, and the colonies were gently scraped from the culture plate. The cells were transferred from the culture dish to a 15 mL centrifuge tube, and 6 mL of N2B27 complete medium was added to bring the total volume of the cell suspension to 8 mL. Gently mix the cell suspension to break up large cells. Transfer 1 mL of the cell suspension to the prepared coated petri dish. Transfer the newly plated cells to the incubator, gently rock the plate back and forth and evenly distribute it. The cells were incubated overnight in a 37 ° C tissue incubator.
3、iPS细胞悬浮培养及向皮肤类器官的诱导分化3, iPS cell suspension culture and induced differentiation into skin organs
诱导iPSC细胞向3个胚层细胞和传代培养需要在生物安全柜中进行。在传代后的第二 天观察细胞状态,以确认iPSC的贴壁情况。如果iPSCs开始形成克隆,将继续下面的分化实验。开始时分化时为第0天,在第0天将iPSCs用1X TrypLE Express酶(GIBCO)或Accutase细胞解离试剂(GIBCO)解离,重悬于外胚层分化培养基中,并接种在96孔低细胞粘附性U底培养板上,每孔为100μL,每孔3×10 3个细胞。在第1天,将每孔中50μL(一半)培养基移除并用50μL含有4%(v/v)Matrigel(终浓度2%;Corning)的新鲜外胚层分化培养基补充。在第3天,向每个孔中加入25μL含有50ng/mL BMP-4(PeproTech)和5μM SB431542(Stemgent)的新鲜外胚层分化培养基(无Matrigel),使最终浓度为10ng/mL BMP-4和1μM SB431542,最终体积为125μL/孔。在不同的情况下,BMP-4的工作浓度增加至50ng/mL的最终浓度,这在质量上改善了皮肤类器官诱导效率。在第4天,向各孔中加入25μL含有6μM LDN(Stemgent)和150ng/mL FGF-2(PeproTech)的新鲜外胚层分化培养基(无Matrigel),终浓度为1μM LDN和25ng/mL FGF-2,此时培养液的最终体积150μL/孔。在第8天,在含有1%(v/v)Matrigel的500μL成熟培养基中的24孔低细胞粘附板(Nunclon Sphera)上将每个细胞聚集体转移至单个孔中。从第10天开始,每隔一天将的培养基(250μL)的一半去除并补充250μL新鲜成熟培养基(不含Matrigel)直到第30天。 Induction of iPSC cells into 3 germ layer cells and subculture is performed in a biosafety cabinet. The state of the cells was observed on the second day after passage to confirm the adherence of iPSC. If iPSCs begin to form clones, the following differentiation experiments will continue. Initially, the day of differentiation was day 0. On day 0, iPSCs were dissociated with 1X TrypLE Express enzyme (GIBCO) or Accutase cell dissociation reagent (GIBCO), resuspended in ectodermal differentiation medium, and seeded in 96 wells. On a low cell adhesion U-bottom plate, 100 μL per well, 3 × 10 3 cells per well. On day 1, 50 [mu]L (half) of the medium in each well was removed and supplemented with 50 [mu]L of fresh ectodermal differentiation medium containing 4% (v/v) Matrigel (final concentration 2%; Corning). On day 3, 25 μL of fresh ectodermal differentiation medium (without Matrigel) containing 50 ng/mL BMP-4 (PeproTech) and 5 μM SB431542 (Stemgent) was added to each well to give a final concentration of 10 ng/mL BMP-4. And 1 μM SB431542, final volume 125 μL/well. In different cases, the working concentration of BMP-4 was increased to a final concentration of 50 ng/mL, which improved the skin-like organ induction efficiency in quality. On day 4, 25 μL of fresh ectodermal differentiation medium (without Matrigel) containing 6 μM LDN (Stemgent) and 150 ng/mL FGF-2 (PeproTech) was added to each well to a final concentration of 1 μM LDN and 25 ng/mL FGF- 2. At this time, the final volume of the culture solution was 150 μL/well. On day 8, each cell aggregate was transferred to a single well on a 24-well low cell adhesion plate (Nunclon Sphera) in 500 [mu]L of maturation medium containing 1% (v/v) Matrigel. Starting from day 10, half of the medium (250 μL) was removed every other day and supplemented with 250 μL of fresh mature medium (without Matrigel) until day 30.
实施例3Example 3
1、细胞移植和毛发形成及毛囊的分离1. Cell transplantation and hair formation and separation of hair follicles
利用诱导多能干细胞形成毛囊结构,具体过程如下:(1)培养实施例2中获得的已经部分分化的诱导多能干细胞,将其用Accutase酶消化后,制备总细胞数200万的细胞悬液于PBS中,300g离心5min。(2)取离心后的细胞,弃上清,在细胞沉淀中加入50微升Geltrex,混匀,待用,同时加入Wnt10b因子,使其在Geltrex中的浓度是1ug/ml。(3)将怀孕60天左右的胚胎猪通过剖腹手术从母猪腹腔中取出,但不让猪胎儿脱离母体,用打孔器在其背部皮肤上制造面积约7mm 2的伤口。(4)将步骤(2)中制备获得的混有皮肤类器官细胞的Geltrex移植到胚胎猪皮肤伤口表面。(5)用3M膜覆盖胚胎猪伤口表面,然后将胚胎猪放置回到母体羊膜内,缝合羊膜(参考图3)。(6)正常饲养怀孕母猪,正常分娩后小猪1-6个月,切取细胞移植的皮肤组织,并固定染色后,观察毛囊结构,分析毛囊再生情况(参考图4)。 The hair follicle structure is formed by using induced pluripotent stem cells, and the specific process is as follows: (1) The partially differentiated induced pluripotent stem cells obtained in Example 2 are cultured, and after being digested with Accutase, a cell suspension having a total cell count of 2 million is prepared. Centrifuge at 300 g for 5 min in PBS. (2) The cells after centrifugation were taken, and the supernatant was discarded. 50 μl of Geltrex was added to the cell pellet, mixed, and used, and Wnt10b factor was added to make the concentration in Geltrex 1 ug/ml. (3) The embryo pigs aged about 60 days were removed from the sow's abdominal cavity by laparotomy, but the pig fetus was not removed from the mother body, and a wound of about 7 mm 2 was made on the back skin with a puncher. (4) The Geltrex mixed with the skin-like organ cells obtained in the step (2) is transplanted to the surface of the embryonic pig skin wound. (5) Cover the surface of the embryonic pig wound with a 3M membrane, then place the embryo pig back into the maternal amniotic membrane and suture the amnion (refer to Figure 3). (6) Normally rearing pregnant sows, piglets after normal delivery for 1-6 months, cut the skin tissue of the transplanted cells, and after fixed staining, observe the structure of hair follicles and analyze the regeneration of hair follicles (refer to Figure 4).
2、再生毛囊的形态学鉴定及细胞来源分析2. Morphological identification and cell source analysis of regenerated hair follicles
对实施例3和实施例4中实验组和对照组的干细胞移植的皮肤组织进行组织形态学鉴定,具体如下:制备表皮干细胞再生毛囊结构组织切片:(1)分别切取实施例3和实施例4中实验组和对照组的干细胞移植的皮肤组织区域,4%多聚甲醛固定过夜,PBS浸泡3小时,30%蔗糖脱水8小时。(2)OCT包埋组织,冰冻切片,取10μm厚度的组织切片。(3)将组织切片自然晾干。(4)将组织切片用PBS洗三遍,每次5分钟。(5)用0.2-0.5%triton X-100 (PBS配制)对组织切片做透膜处理10分钟。(6)将透膜处理后的组织切片用PBS洗三遍,每次5分钟。(7)将组织切片用2%BSA封闭30分钟,然后用PBS洗两遍。(8)将封闭后的组织切片加入抗生物素Cy3标记的1抗,室温孵育3小时。(9)将连接1抗的组织切片用PBS洗三遍,每次5分钟。(10)加入0.5μg/ml DAPI(PBS配制)染色10分钟。(11)将染色后组织切片用PBS洗三遍,去除多余的DAPI。(12)将去除多余的DAPI的组织切片加入20μl封片剂封片。(13)封片好的组织染色样品置于4度冰箱中,可存放2周以上。组织切片的组织形态学鉴定:采用共聚焦显微镜观察组织切片,观察发现,实施例2的再生皮肤组织中有毛囊结构的形成,其结构形态与正常皮肤中的毛囊类似;同时人源细胞特异性标记MAB1281染色结果显示,再生毛囊的细胞都显示MAB1281阳性,证明新生的毛囊是从移植的人多能性干细胞分化产生的(结果参考图5)。The histomorphological identification of the stem tissue transplanted skin tissue of the experimental group and the control group in Example 3 and Example 4 was as follows: Preparation of epidermal stem cell regeneration hair follicle structure tissue section: (1) Excision Example 3 and Example 4, respectively The skin tissue area of the stem cell transplantation of the middle experimental group and the control group was fixed with 4% paraformaldehyde overnight, immersed in PBS for 3 hours, and dehydrated with 30% sucrose for 8 hours. (2) OCT embedded tissue, frozen sections, and tissue sections of 10 μm thickness were taken. (3) Dry the tissue section naturally. (4) Tissue sections were washed three times with PBS for 5 minutes each time. (5) Tissue sections were diafiltered for 10 minutes with 0.2-0.5% triton X-100 (in PBS). (6) The tissue sections after the membrane treatment were washed three times with PBS for 5 minutes each time. (7) Tissue sections were blocked with 2% BSA for 30 minutes and then washed twice with PBS. (8) The blocked tissue sections were added to the avidin Cy3 labeled 1 antibody and incubated for 3 hours at room temperature. (9) The tissue sections to which the primary antibody was ligated were washed three times with PBS for 5 minutes each time. (10) Dyeing was carried out by adding 0.5 μg/ml DAPI (in PBS preparation) for 10 minutes. (11) The stained tissue sections were washed three times with PBS to remove excess DAPI. (12) Tissue sections from which excess DAPI was removed were added to a 20 μl capsule seal. (13) The tissue dyeing sample with good sealing is placed in a 4 degree refrigerator and can be stored for more than 2 weeks. Histomorphological identification of tissue sections: Tissue sections were observed by confocal microscopy. It was observed that the hair follicle structure was formed in the regenerated skin tissue of Example 2, and its structural morphology was similar to that of normal skin hair follicles; The staining results of the labeled MAB1281 showed that the cells of the regenerated hair follicles all showed MAB1281 positive, indicating that the newborn hair follicles were differentiated from the transplanted human pluripotent stem cells (results refer to Figure 5).
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification, as well as features of various embodiments or examples, may be combined and combined.
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims (12)

  1. 一种获得人工毛囊的方法,其特征在于,包括:A method for obtaining artificial hair follicles, comprising:
    将人多能干细胞移植入异种胚胎的皮肤中,所述异种胚胎处于毛发形成阶段;Transplanting human pluripotent stem cells into the skin of a heterologous embryo, the heterogeneous embryo being in a stage of hair formation;
    将移植有人多能干细胞的胚胎在异种母体内继续进行培养,直至胚胎娩出;以及An embryo transplanted with a pluripotent stem cell is continuously cultured in a heterologous mother until the embryo is delivered;
    分离胚胎中移植有人多能干细胞的皮肤中的毛囊,以便获得所述人工毛囊。The hair follicles in the skin of the pluripotent stem cells are transplanted in the embryos to obtain the artificial hair follicles.
  2. 根据权利要求1所述的方法,其特征在于,所述人多能干细胞为诱导多能干细胞(iPSCs),所述移植之前进一步包括:将所述诱导多能干细胞分化为皮肤类器官,所述皮肤类器官包括外胚层细胞,中胚层细胞和神经内胚层细胞;The method according to claim 1, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs), and the transplanting further comprises: differentiating the induced pluripotent stem cells into skin-like organs, Skin-like organs include ectoderm cells, mesoderm cells, and neuroectodermal cells;
    任选地,所述诱导多能干细胞来源于血液细胞或皮肤细胞。Optionally, the induced pluripotent stem cells are derived from blood cells or skin cells.
  3. 根据权利要求2所述的方法,其特征在于,将所述诱导多能干细胞分化为皮肤类器官包括:The method of claim 2 wherein the differentiating said induced pluripotent stem cells into dermatophyte comprises:
    将所述诱导多能干细胞进行悬浮培养,以便得到3D细胞球;The induced pluripotent stem cells are subjected to suspension culture to obtain a 3D cell sphere;
    将所述3D细胞球进一步进行分化培养,以便获得皮肤类器官。The 3D cell spheres are further subjected to differentiation culture to obtain a skin-like organ.
  4. 根据权利要求2所述的方法,其特征在于,将所述诱导多能干细胞分化为皮肤类器官是在所述多能干细胞与BMP-4,SB431542,FGF-2和LND接触的条件下进行的;The method according to claim 2, wherein the differentiation of the induced pluripotent stem cells into dermatophytes is carried out under conditions in which the pluripotent stem cells are in contact with BMP-4, SB431542, FGF-2 and LND. ;
    优选地,所述BMP-4的浓度为50ng/mL。Preferably, the concentration of BMP-4 is 50 ng/mL.
  5. 根据权利要求4所述的方法,其特征在于,进一步包括将所述皮肤类器官进行消化处理,以便获得单细胞混合液;The method according to claim 4, further comprising digesting said skin-like organ to obtain a single-cell mixture;
    任选地,将所述单细胞混合液与基质胶或凝胶类物质混合;Optionally, mixing the single cell mixture with a matrigel or gel-like substance;
    任选地,所述基质胶包括选自Geltrex和ColI的至少之一。Optionally, the Matrigel comprises at least one selected from the group consisting of Geltrex and ColI.
  6. 根据权利要求1所述的方法,其特征在于,所述人多能干细胞包括选自毛囊干细胞、皮肤干细胞及其它具有毛囊形成或诱导能力的干细胞。The method of claim 1 wherein said human pluripotent stem cells comprise stem cells selected from the group consisting of hair follicle stem cells, skin stem cells, and other hair follicle forming or inducing abilities.
  7. 根据权利要求1所述的方法,其特征在于,所述人多能干细胞包括基因修饰的多能干细胞或诱导多能干细胞;The method according to claim 1, wherein said human pluripotent stem cells comprise genetically modified pluripotent stem cells or induced pluripotent stem cells;
    任选地,所述基因修饰为基因敲除或基因表达。Optionally, the genetic modification is gene knockout or gene expression.
  8. 根据权利要求1所述的方法,其特征在于,所述移植之前进一步包括:将所述人多能干细胞与促进毛囊形成的因子混合;The method according to claim 1, wherein said transplanting further comprises: mixing said human pluripotent stem cells with a factor that promotes hair follicle formation;
    任选地,所述促进毛囊形成的因子为Wnt10b因子;Optionally, the factor that promotes hair follicle formation is a Wnt10b factor;
    任选地,所述Wnt10b因子的浓度为1ug/mL。Optionally, the concentration of the Wnt10b factor is 1 ug/mL.
  9. 根据权利要求1所述的方法,其特征在于,所述异种胚胎包括选自猪胚胎、灵长类动物胚胎、兔胚胎、牛胚胎以及羊胚胎的至少之一。The method according to claim 1, wherein the heterologous embryo comprises at least one selected from the group consisting of a pig embryo, a primate embryo, a rabbit embryo, a bovine embryo, and a sheep embryo.
  10. 根据权利要求1所述的方法,其特征在于,所述分离是在胚胎分娩后的第0~6个月内进行的。The method of claim 1 wherein said separating is performed within 0 to 6 months after delivery of the embryo.
  11. 一种人工毛囊,其特征在于,是由权利要求1~10任一项所述的方法获得的。An artificial hair follicle obtained by the method according to any one of claims 1 to 10.
  12. 一种毛囊种植的方法,其特征在于,将权利要求11所述的人工毛囊接种到自体皮肤表面。A method of growing hair follicles, characterized in that the artificial hair follicle of claim 11 is inoculated onto the surface of the autologous skin.
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