WO2019148395A1

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

Info

Publication number: WO2019148395A1
Application number: PCT/CN2018/074827
Authority: WO
Inventors: 王旭升; 吴耀炯; 牟丽莎; 曹楠
Original assignee: 深圳市祥云生物科技有限公司
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2019-08-08

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

Artificial hair follicle and preparation method and application thereof

Technical field

The present invention relates to the field of biotechnology, and in particular, to artificial hair follicles and methods for their preparation and use.

Background technique

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.

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.

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 (

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.

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.

Summary of the invention

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.

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.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

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.

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.

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.

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.

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.

According to an embodiment of the present invention, the transplanting further comprises: digesting the skin-like organ to obtain a single-cell mixture.

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.

According to an embodiment of the invention, the Matrigel comprises at least one selected from the group consisting of Geltrex and ColI.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

DRAWINGS

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;

2 is a diagram showing induced differentiation of iPS cells into skin cells according to an embodiment of the present invention,

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;

3 is a mixture of differentiated iPS cells and a gel, and transplantation of cells according to an embodiment of the present invention;

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.

Detailed ways

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.

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.

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.

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.

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.

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.

Example 1

1. Preparation, induction and detection of peripheral blood cells of the cell provider:

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. Method for inducing pluripotent cell differentiation in vitro by iPS

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. The karyotype analysis process of induced pluripotent cells

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.

Example 2

1. Preparation of iPS cells to skin-like organs for trophoblast-free culture

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. Preparation before induction of iPS cells.

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 cell suspension culture and induced differentiation into skin organs

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.

Example 3

1. Cell transplantation and hair formation and separation of hair follicles

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. Morphological identification and cell source analysis of regenerated hair follicles

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).

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.

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

A method for obtaining artificial hair follicles, comprising:

Transplanting human pluripotent stem cells into the skin of a heterologous embryo, the heterogeneous embryo being in a stage of hair formation;

An embryo transplanted with a pluripotent stem cell is continuously cultured in a heterologous mother until the embryo is delivered;

The hair follicles in the skin of the pluripotent stem cells are transplanted in the embryos to obtain the artificial hair follicles.
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;

Optionally, the induced pluripotent stem cells are derived from blood cells or skin cells.
The method of claim 2 wherein the differentiating said induced pluripotent stem cells into dermatophyte comprises:

The induced pluripotent stem cells are subjected to suspension culture to obtain a 3D cell sphere;

The 3D cell spheres are further subjected to differentiation culture to obtain a skin-like organ.
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. ;

Preferably, the concentration of BMP-4 is 50 ng/mL.
The method according to claim 4, further comprising digesting said skin-like organ to obtain a single-cell mixture;

Optionally, mixing the single cell mixture with a matrigel or gel-like substance;

Optionally, the Matrigel comprises at least one selected from the group consisting of Geltrex and ColI.
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.
The method according to claim 1, wherein said 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.
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;

Optionally, the factor that promotes hair follicle formation is a Wnt10b factor;

Optionally, the concentration of the Wnt10b factor is 1 ug/mL.
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.
The method of claim 1 wherein said separating is performed within 0 to 6 months after delivery of the embryo.
An artificial hair follicle obtained by the method according to any one of claims 1 to 10.
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.