CN110093306A - Artificial hair follicle and its preparation method and application - Google Patents
Artificial hair follicle and its preparation method and application Download PDFInfo
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- CN110093306A CN110093306A CN201810094681.0A CN201810094681A CN110093306A CN 110093306 A CN110093306 A CN 110093306A CN 201810094681 A CN201810094681 A CN 201810094681A CN 110093306 A CN110093306 A CN 110093306A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/10—Hair or skin implants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
- A61B2017/00743—Type of operation; Specification of treatment sites
- A61B2017/00747—Dermatology
- A61B2017/00752—Hair removal or transplantation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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Abstract
The invention proposes a kind of methods for obtaining artificial hair follicle.This method comprises: human pluripotent stem cells are implanted into the skin of mutant embryos, the mutant embryos are in hair formation stages；There is the embryo of human pluripotent stem cells to continue to cultivate in xenogenesis parent transplanting, until embryo gives birth to；And transplanting has the hair follicle in the skin of human pluripotent stem cells in separation embryo, to obtain the artificial hair follicle.The artificial hair follicle quantity obtained according to the method for the embodiment of the present invention is more, and structural form is similar with the hair follicle in normal skin, can be used for the hair transplantation of people's different parts, such as scalp, eyebrow, beard, chaeta；It is not in immunological rejection that the artificial hair transplantation obtained simultaneously, which returns to multipotential stem cell supplier, and safety greatly improves.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to artificial hair follicle and its preparation method and application.
Background technique
It is strong to have become the increasingly severe Asia of the modern male's (including some women) of puzzlement for exiguous human hair caused by alopecia
Kang Wenti, although alopecia will not serious physiological health be topic, alopecia can it is serious influence individual psychological condition, so
Serious it can reduce personal quality of life.According to incompletely statistics, China has more than 200,000,000 people at present there are different degrees of alopecias
Problem, in the whole world, such crowd's body alreadys exceed 1,000,000,000.Pathologic exception epilation, it is bald such as male, be it is certain because
Hair follicle is chronically at stand-down and cannot be introduced into growth cycle under the action of element, is clinically directed to the drug of pathologic alopecia now
It is very few, and these drugs are also to have effect, such as minoxidil and Finasteride just for the patient of seldom ratio, are all faced
Two kinds of most widely used Anti-hair loss drugs of bed, are that two kinds of drugs will not only be taken for a long time, have different degrees of side effect, simultaneously
Anticreep effect is also fainter.
Stem cell (stem cells) is human body and its various histiocytic primary sources, most significant biological characteristics
Sign is existing self-renewing and the ability that is constantly proliferated, and has the potential of Multidirectional Differentiation.Stem cell is divided into according to different sources
Adult stem cell (somaticstem cells) and embryonic stem cell (embryonic stem cells, ES cell).At soma
Cell includes mesenchymal stem cell, pancreatic stem cells, neural stem cell etc., present in adult tissue.2006, day
This Kyoto University Zhong Shan stretches more (Yamanaka) research group and uses outer-gene rotaring dyeing technology, filters out from 24 factors
Above-mentioned 4 transcription factors are imported fetal mice by retrovirus by 4 transcription factors such as Oct4, Sox2, c-Myc, Klf4
Fibroblast or adult mice tail skin fibroblast, obtain under the condition of culture of mouse ES (embryonic stem cell) cell
The pluripotent stem cell system of Fbx15+ was obtained, this kind of iPS cell is in cellular morphology, proliferative capacity, surface antigen marker, gene table
It is similar to hES cell up to spectrum, the epigenetics state of pluripotent stem cell specific gene, telomerase activation etc., and
And when cultivating in vitro and in Mice Body teratoma formed in can be divided into the different cell types of 3 germinal layers
(Takahashi K et al.Cell 2007；131:861-872).This is known as the weight of bioscience " milestone " by educational circles
Quantum jump is expected to the ethics helped scientist around clone technology, morals dispute, opens gate for medical application.
Since mid-term the 1990s, oval scalp cutting acquisition is to obtain the first choice of hair transplantation hair follicle colony
Method.In past 20 years, Follicular Unit extracts the method that (FUE) has become the acquisition donor hair being becoming increasingly popular
(Rassman,Bernstein et al.2002,Facial Plast Surg Clin North Am,Harris 2013,
Dermatol Surg.).FUE obtains independent Follicular Unit (Cole from donor areas using manual or robot device
2013, Facial Plast Surg Clin North Am.), FUE is no confession relative to the major advantage of oval donor harvesting
Body hair harvest then sutures or the linear scar of sewn closed.This is a very big advantage, short especially for hair
Law-executor will not see apparent scar.Although FUE has the advantages that without linear scar, however it remains otherwise
Problem and potential long-term side-effects.As full-thickness incision, a 1mm diameter can be generated by harvesting each Follicular Unit
The scar of formed punch.These scars can coloring to remaining hair follicle and development have an impact.In addition, no matter FUE be by manual, it is motor-driven
Or robot punching progress, it cuts and is harvested as hair follicle with donor ellipse scalp, it is obvious to there is the hair from donor areas
The risk of consumption, the i.e. hair of donor areas can detail reductions.This may generate iatrogenic " moth food " or " pseudo- syphilis " appearance ( N et al.J Plast Reconstr Aesthet Surg.2012,Poswal A et al.Indian J
Dermatol.2011).FUE is the effective and useful mode for obtaining donor hair.It, which is generated, cuts donor than oval scalp
Hair follicle acquisition can generate few visible scar.As any surgical technic, there are limitations and side effect for this method.
In short, hair transplantation becomes the scheme for solving the problems, such as alopecia emerging in recent years in addition to drug therapy.But hair follicle
The method of transplanting is after taking the hair follicle of rear pillow part, then by the position of the forehead exiguous human hair of the hair transplantation to the same person, though
Right this method is got instant result, and is taken effect quickly, but be after all as robbed Peter to pay Paul, it is only effective to the patient of a small amount of alopecia,
But for the therapeutic effect that the patient of large area alopecia can not have.Hair follicle extraction simultaneously is that mechanicalness damage can be generated to hair follicle
Wound, influences the survival rate of hair follicle.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, the present invention mentions
A kind of hair follicle regeneration method based on people's autologous stem cells and xenogenesis carrier is gone out.
In the first aspect of the present invention, the invention proposes a kind of methods for obtaining artificial hair follicle.Reality according to the present invention
Apply example, which comprises human pluripotent stem cells are implanted into the skin of mutant embryos, the mutant embryos are in hair shape
At the stage；There is the embryo of human pluripotent stem cells to continue to cultivate in xenogenesis parent transplanting, until embryo gives birth to；And point
There is the hair follicle in the skin of human pluripotent stem cells from transplanting in embryo, to obtain the artificial hair follicle.Implement according to the present invention
The artificial hair follicle quantity that the method for example obtains is more, and structural form is similar with the hair follicle in normal skin, can be used for people's different parts
Hair transplantation, such as scalp, eyebrow, beard, chaeta；The artificial hair transplantation obtained simultaneously returns to multipotential stem cell supplier
It is not in immunological rejection, safety greatly improves.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, the human pluripotent stem cells are to induce multi-potent stem cell (iPSCs), the transplanting
Taking a step forward includes: that described induce multi-potent stem cell is divided into skin organoid, and the skin organoid includes that ectoderm is thin
Born of the same parents, mesoblastema and neural endoderm cell.Stem cell will be divided into 3 germinal layers first, further be divided into skin
Organ, to further be divided into hair follicle structure.
According to an embodiment of the invention, inducing multi-potent stem cell that be divided into skin organoid include: to lure described for described
It leads multipotential stem cell and carries out suspension culture, to obtain 3D cell ball；The 3D cell ball further progress is broken up and is cultivated, with
Just skin organoid is obtained.
The source that induces multi-potent stem cell is not particularly limited, and can be derived from any one adult cell.According to this hair
Bright embodiment, it is described to induce multi-potent stem cell from blood cell or Skin Cell.
According to an embodiment of the invention, inducing multi-potent stem cell that be divided into skin organoid done in the multipotency for described
It is carried out under conditions of cell and BMP-4, SB431542, FGF-2 and LND contact.
According to an embodiment of the invention, the concentration of the BMP-4 is 50ng/mL.And then further improve skin device
The induced efficiency of official.
According to an embodiment of the invention, further comprising before the transplanting: the skin organoid is carried out at digestion
Reason, to obtain unicellular mixed liquor.
According to an embodiment of the invention, the unicellular mixed liquor is mixed with matrigel or gel-like substance.And then institute
Membranaceous cellular layer can be formed under the induction for the factor for promoting hair follicle to be formed by stating unicellular mixed liquor.Implementation according to the present invention
Example, the thickness of the membranaceous cellular layer are identical as the skin thickness of receptor mutant embryos.
According to an embodiment of the invention, the matrigel includes being selected from least one of Geltrex and ColI.
According to an embodiment of the invention, the human pluripotent stem cells include selected from hair follicle stem cells, skin progenitor cell and its
It has the stem cell of hair follicle formation or inducibility.
According to an embodiment of the invention, the human pluripotent stem cells include the multipotential stem cell or induced multi-potent of gene modification
Stem cell.Optionally, the gene modification is gene knockout or gene expression.
According to an embodiment of the invention, further comprising before the transplanting: by the human pluripotent stem cells and promoting hair
The factor mixing that capsule is formed.And then hair follicle formation efficiency greatly improves.
According to an embodiment of the invention, the factor for promoting hair follicle to be formed is the Wnt10b factor.And then hair follicle forms effect
Rate further increases.
According to an embodiment of the invention, the concentration of the Wnt10b factor is 1ug/mL.And then hair follicle formation efficiency is into one
Step improves.
According to an embodiment of the invention, the mutant embryos include selected from Pig embryos, primate embryonic, rabbit embryonic,
At least one of ox embryo and sheep embryo.It should be noted that the heterogenous animal carrier must reach clinical when in use
Sanitary standard can neither carry any clinical clearly specified pathogen with pathogenic risk.It should be further noted that
Only develop into hair follicle for human stem cell provides environment to the mutant embryos, needs when being finally separating the artificial hair follicle reached maturity
Completely remove all animal derived tissues and cell.
According to an embodiment of the invention, the separation is carried out in embryo puerperal 0th~6 month.At this time
Hair follicle has more complete structure, has transplanting condition and power of regeneration is strong.
According to an embodiment of the invention, further comprising that obtained artificial hair transplantation is returned to stem cell supplier's skin
Afterwards, whether can survive after judging the hair transplantation and have normal growth cycle.If hair follicle is in one growth period 3 months
Above, while growth period can be entered in 1-3 months after entering stand-down, that is, it survives and has normal after judging the hair transplantation
Growth cycle.It can be used to subsequent hair transplantation.
In the second aspect of the present invention, the invention proposes a kind of artificial hair follicles.According to an embodiment of the invention, the hair
Capsule is obtained by mentioned-above method.Artificial hair follicle according to an embodiment of the present invention can be removed from entire skin histology
Out, the damage of hair follicle can utmostly be reduced；Artificial hair follicle according to an embodiment of the present invention is that autologous stem cells produce simultaneously
Raw self hair follicle is not in immunological rejection, safety is more if these hair transplantations are returned to stem cell supplier
It is high.
In the third aspect of the present invention, the invention proposes a kind of methods of hair follicle plantation.According to an embodiment of the invention,
The method includes mentioned-above artificial hair follicle is inoculated into autologous skin surface.It can answer according to the method for the embodiment of the present invention
The hair follicle that hair follicle plantation, hair encryption for male pattern baldness (getting a bit thin on top) use is planted, the hair follicle that hair line adjustment uses is planted,
The hair follicle of cicatrical scalp is planted, and also includes that eyebrow is planted, beard plantation and chaeta plantation.
It should be noted that " transplanting " of the present invention, " inoculation " operation, are not particularly limited, those skilled in the art
Member can be easily accomplished, and be not required to professional training.
Detailed description of the invention
Fig. 1 is according to an embodiment of the present invention based on the hair follicle regeneration method induced multi-potent stem cell self with xenogenesis carrier
Flow chart；
Fig. 2 is induction differentiation figure of the iPS cell according to an embodiment of the present invention to Skin Cell,
Wherein, a, the isolated mononuclearcell from blood；Mononuclearcell induction is become iPS cell, iPS by b
Cell forms clone；C, iPS cell break up to skin keratinocytes；
Fig. 3 is that the iPS cell of differentiation according to an embodiment of the present invention mixes and the transplanting of cell with gel；
Fig. 4 is the result figure of the stem cell formation hair follicle structure after transplanting according to an embodiment of the present invention；And
Fig. 5 be the hair follicle that human archeocyte specific marker MAB1281 according to an embodiment of the present invention dyeing display is formed and
Result figure of the skin texture entirely from source of people stem cell.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
The hair follicle regeneration method based on people's autologous stem cells and xenogenesis carrier that the present invention provides a kind of, wherein hair follicle is again
Raw method includes the separation, culture and induction of human body cell or stem cell, wherein there are two ways to obtaining stem cell, first
Kind is the hair follicle stem cells or Skin Cell for being directly separated hair loss patient；Second is by adult cell (such as haemocyte and skin
Cell) as the versatile stem cell with Multidirectional Differentiation potentiality, which is that (iPS is thin for inductive pluripotent stem cells for induction
Born of the same parents), i.e. acquisition hair loss patient or the person that needs hair transplant induces multi-potent stem cell, which has had effective realization at present
Means.Ectoderm cell is divided into after being induced multi-potent stem cell first, then is mixed with matrigel or gel-like substance,
Simultaneously be added can promote ectoderm cell to hair follicle break up and develop the factor, allow induction after cell and gel-like bracket substance
Form membranaceous cellular layer.The thickness of the membranaceous cellular layer should be identical with receptor heterogenous animal fetal skin thickness.At this time due to
Hair follicle in receptor fetal skin is in generation and stage of development, so its microenvironment is most suitable for hair follicle regeneration, so
Being divided into ectodermic human stem cell and can effectively form hair under the action of hair follicle differentiation induction factor again after transplanting
Capsule.For hair follicle stem cells, the induction of outside differentiation of germinal layers, but the effect in order to promote hair follicle to be formed are not needed then before transplantation
Rate, there is still a need for mix before transplantation with the factor for promoting hair follicle to be formed.
When carrier animal fetal birth, individual developmental is at hair follicle for the human stem cell transplanted in skin, at this time
Hair follicle there is more complete structure, part hair follicle has had hair to grow.Hair follicle at this time has been provided with transplanting item
Part, it is possible to just separate human body hair follicle in carrier animal fetal birth, then plant.It can also go out in carrier animal fetus
Raw raising a period of time, the human body hair follicle for allowing it to carry further are reached maturity, and then separate plantation again.Xenogenesis carrier animal
Including different cultivars pig, primate and rabbit, ox, sheep.It is contemplated that ethics and research background and safety, reach medical treatment
The pig of grade health level should be the animal preferentially used.It is all to be reached using heterogenous animal carrier in practical application
Clinical sanitary standard can neither carry any clinical clearly specified pathogen with pathogenic risk.For pig, to make
With the strain for not carrying endogenous retrovirus.
There are many practical application directions by the artificial hair follicle of the invention, including for treating male pattern baldness (getting a bit thin on top)
Hair follicle plantation, for the hair follicle plantation that hair encryption uses, for the hair follicle plantation that hair line adjustment uses, for cicatrical
The hair follicle of scalp is planted, and also includes that eyebrow is planted, beard plantation and chaeta plantation.Since the characteristics of invention is by from soma
The completely new hair follicle of cytothesis, so it has the advantage of several respects in application process, firstly, after passing through after culture
Stem cell or inductivity versatile stem cell can quantitatively massive amplification, so theoretically this method can obtain arbitrarily
The hair follicle of quantity；Different growth factors can be added in stem cell stage of development in carrier animal, and control hair follicle growth is not
Same size and shape, for the hair follicle regeneration of different parts, such as eyebrow and beard；Hair follicle is separated in the present invention can be from whole
Hair follicle is removed in a skin histology, so as to utmostly reduce the damage to hair follicle；The regenerated hair follicle of the present invention is self
The self hair follicle that stem cell generates, so being not in that immunological rejection is anti-these hair transplantations are returned to stem cell supplier
It answers.
In order to make it easy to understand, according to an embodiment of the present invention based on the hair follicle induced multi-potent stem cell self with xenogenesis carrier
Regeneration method flow chart refers to Fig. 1.
Specific embodiments of the present invention are described below in detail, examples of the embodiments are shown in the accompanying drawings.Below by
The embodiment being described with reference to the drawings is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
Embodiment 1
1, the peripheral blood cells of cell supplier prepare, induction and detection:
30ml peripheral blood is isolated by vein haemospasia from cell donor person, passes through Ficoll-Hypaque density gradient
Separation obtains monokaryon (MNC) cell.MNC cell is being contained into 10%FBS and 10ng/ml IL-7 or 10ng/ml G-CSF, GM-
It is cultivated 5 days in the α-MEM culture medium of CSF, IL-3 and IL-6.These subsequent cells have for generating inductive pluripotent stem cells
Steps are as follows for body, by 1-2 × 104A MNC is placed in 1 hole of 96 orifice plates, and reprograms kit with CytoTune-iPS
(DNAVEC company, Tsukuba, Japan) infection coding OCT4, SOX2, KLF4, cMYC F- deficiency sendai virus vector
(Sev/ Δ F), wherein the infection multiple (MOI) of each factor is 5-10.One day after, low-speed centrifugal removes culture solution for infection, receives
Cell is obtained, and is cultivated in six orifice plates with mouse embryonic fibroblasts (MEF) shop fixtures culture, in used medium
It is identical as above-mentioned culture medium, it cultivates other one day.2nd day, culture medium is changed to the people ES (people for being supplemented with 8ng/ml bFGF
Embryonic stem cell) culture medium, and liquid is changed with fresh culture daily.Morphological category is similar to ES clone the 13rd day or so after infection
Start to occur；In the 21st or 28 day picked clones, expand culture, and versatility is checked by immunofluorescence or flow cytometry
Label.Wherein the method for the detection pluripotency marker of immunofluorescence is that iPS cell is washed 3 times with D-PBS and is existed at room temperature
10 minutes are fixed in 4% paraformaldehyde.After being washed with D-PBS, made cell rupture of membranes (room temperature 1 minute) with methanol, D-PBS is washed simultaneously
It is closed 30 minutes in PBS-5% donkey serum.By anti-Oct4 (mouse monoclonal), Tra1-81 (mouse monoclonal) and Nanog's
Antibody is stayed overnight in 4 DEG C of incubation incubated cells.Cell is washed 3 times with D-PBS, and carries out suitable fluorescence secondary antibody at room temperature
(Invitrogen) 1 hour, then DAPI was dyed 1 minute.Pluripotency marker's expression is detected under fluorescence microscope.Monster
The method of tumor experiment is the iPS cell of blood sources to be collected with collagen enzymatic treatment (1mg/ml), and settling by cell aggregation will
IPS cell is separated from trophocyte.Cell is washed with D-PBS again, is resuspended in 500 μ l people's ES cell culture mediums and subcutaneous
It is injected into SCID mice (Taconic).After injection 4-6 weeks, tumour is taken out from the mouse of euthanasia, and in 4% poly
It is fixed in formaldehyde.It is dyed according to h and E, by sample paraffin embedding, slice analysis.
2, the method for iPS induced multi-potent cells in vitro differentiation
With 1mg/ml clostridiopetidase A IV (Invitrogen) processing iPSC clone 10 minutes, washed with D-PBS, by scrape and
Liquid relief is dissociated into agglomerate several times.IPSC block is applied to Lipidure clad plate (NOF in the ES culture medium without bFGF
Corporation, Irvine, CA, the U.S.).Replacement culture medium is primary daily.After suspending culture 8 days, embryoid (EB) is turned
It moves on in the coated plate of gelatin, and be further cultured for 8-10 days in identical culture medium.Then the expression feelings of cell marking are analyzed
Condition.
3, the karyotyping process of induced multi-potent cell
It by one day after people's iPSC secondary culture, exposes cells in the gel of 0.25 μ g/ml 3.5 hours, digests,
It collects and is exposed in hypotonic solution (0.4% sodium citrate: 0.4%KCl=1:1) 16 minutes.By cell methanol/acetic acid
(3:1) fixes 2 times, totally 1 hour.Then by cell drop on cold wet and clean sheet glass.Glass slide is incubated for 4 at 70 DEG C
Hour.Then glass slide is handled 10-12 seconds at 37 DEG C with 0.01% trypsase, is washed with 0.9%NaCl, then 37
DEG C with Giemsa solution (Giemsa: phosphate buffer=1.5ml:40ml, pH7.4) dye, 2.5 minutes.Pass through microscope
It checks and determines caryogram.
Embodiment 2
1, non-trophoblast culture from iPS cell to skin Organ Differentiation preparation
Operation needs are carried out in using sterile Biohazard Safety Equipment.Similar with Matrigel, Geltrex matrix is in room
Quickly solidified under warm (RT), it is therefore desirable to the suction nozzle for dispensing by new glue, and being freezed in advance in packing, bracket and
Pipe.50,100 and 200 μ L etc. points of packing is made, and is stored in -80 DEG C.Geltrex is used with 1:100 dilution.Without raising
Layer iPSC culture only needs Geltrex as pan coating agent, however iPSC is broken up, the combination pair of Geltrex and ColI
It is more effective in the differentiation of induction iPS cell to keratinocyte.For 60mm tissue culture dishes Geltrex pan coating process
It is as follows.If it is desired that adjusting accordingly the volume of Geltrex solution with biggish culture dish.50 μ are taken out from -80 DEG C of refrigerators
LGeltrex, and place it in Biohazard Safety Equipment on ice.The cooling sterile DMEM/F12 of 5mL is added into 15mL centrifuge tube.
Using 1mL glass pipet, the cold DMEM/F12 of 1mL is taken from 15mL centrifuge tube, and the Geltrex of freezing is added.Gently up and down
Suction pipe is completely dissolved Geltrex, then the Geltrex of dissolution is transferred to the rest part of the DMEM/F12 in centrifuge tube, mixes
Close diluted Geltrex.50 μ L 3mg/mL ColI stock solutions are added in diluted Geltrex.It will be diluted
Geletrex is mixed with ColI.4 milliliters of above-mentioned solution are added into the culture dish of 60mm.To ensure that whole surface is coated.It will
Culture dish and Geltrex/ColI coating solution are incubated at least 1 hour in 37 DEG C of incubator for tissue cultures.After the completion of coating, by coating
Solution stays in culture dish, and carries out the bed board of iPSCs.Alternatively, drawing coating solution, and the fresh DMEM/F12 of 2mL is added
Enter into coated culture dish.
2, prepare before the induction of iPS cell.
The iPSCs tissue culture dishes for preparing an above-mentioned coating processing 60mm non-trophoblast, are cultivated to about 70% fusion.
Check that cell pollutes and maintain its undifferentiated state to confirm to be not present under the microscope.If cell by environmental stimuli, it
Start to break up, then such cell is not applied to be divided into keratinocyte.Keratinocyte, inventor are divided into for iPSC
The 1:8 of iPSCs is passed on.It is preheated in 37 DEG C of water-baths using N2B27 culture medium and Dispase.Use microscope, confirmation clone
Quantity.Culture medium is gently sucked out from culture dish.2mL 1x PBS is added, PBS is gently sucked out to wash cell in Rotating Plates.
1mL Dispase is added, and culture plate is placed into 37 DEG C of incubator for tissue cultures 3-5 minutes.Geltrex/ is being used by above-mentioned
Geltrex/ColI coating solution (or DMEM/F12) is sucked out in the culture dish of ColI coating procedure, and by the complete N2B27 of 4mL
Culture medium is added in coated culture dish.After iPS cell is incubated for 3-5 minutes with Dispase, microscope confirms around cell clone
Whether curling or folding are had.If digestion is in place, culture plate is transferred to Biohazard Safety Equipment, and Dispase is carefully sucked out.With
After Dispase processing, cell clone is loosely attached to the surface of culture dish very much, if may be shelled using too many power
It falls.The pure DMEM/F12 of 2mL is gently added.Repeated washing 3 times.2mLN2B27 complete medium is added in culture dish, gently
Ground scrapes bacterium colony from culture plate.By cell from being transferred in culture dish in 15mL centrifuge tube, 6mL N2B27 is added and cultivates completely
Base makes the total volume of cell suspending liquid reach 8mL.Cell suspending liquid is gently mixed to be crushed bulk cell.1mL cell is suspended
Liquid is transferred in the coating culture dish prepared.The cell of new bed board is transferred in incubator, lightly wabble board back and forth,
And it is uniformly distributed.Incubated cell is stayed overnight in 37 DEG C of incubator for tissue cultures.
3, iPS cell suspension cultures and to the induction of skin organoid break up
Induction iPSC cell needs to carry out in Biohazard Safety Equipment to 3 endoderm cells and secondary culture.After passage
Second day observation cell state, to confirm the adherent situation of iPSC.If iPSCs initially forms clone, following point will be continued
Change experiment.Be the 0th day when breaking up when beginning, the 0th day by iPSCs 1X TrypLE Express enzyme (GIBCO) or
Accutase cell dissociation reagent (GIBCO) dissociation, is resuspended in ectoderm differential medium, and it is viscous to be seeded in the low cell in 96 holes
On the culture plate of the attached bottom property U, every hole is 100 μ L, every hole 3 × 103A cell.On day 1, by 50 μ L (half) culture medium in every hole
It removes and 50 μ L is used to contain 4% (v/v) Matrigel (final concentration 2%；Corning fresh ectoderm differential medium) is mended
It fills.On day 3,25 μ L are added into each hole and contain 50ng/mL BMP-4 (PeproTech) and 5 μM of SB431542
(Stemgent) fresh ectoderm differential medium (no Matrigel), makes ultimate density 10ng/mL BMP-4 and 1 μM
SB431542, final volume are 125 holes μ L/.In different situations, the working concentration of BMP-4 increases to the final of 50ng/mL
Concentration, this improves skin organoid induced efficiency in quality.On day 4,25 μ L are added into each hole and contain 6 μM of LDN
(Stemgent) and the fresh ectoderm differential medium (no Matrigel) of 150ng/mL FGF-2 (PeproTech), end are dense
Degree is 1 μM of LDN and 25ng/mL FGF-2, at this time 150 hole μ L/ of final volume of culture solution.At the 8th day, containing 1% (v/
V) each cell is gathered on the low cell adherence plate in 24 holes (Nunclon Sphera) in the 500 μ L maturation mediums of Matrigel
Collective is transferred in single hole.Since the 10th day, every other day by the half of culture medium (250 μ L) remove and supplement 250 μ
L fresh mature culture medium (being free of Matrigel) was until the 30th day.
Embodiment 3
1, cell transplantation and hair formation and the separation of hair follicle
Hair follicle structure is formed using inducing multi-potent stem cell, detailed process is as follows: (1) cultivate and obtained in embodiment 2
Inducing multi-potent stem cell through partial differentiation prepares the cell suspension of total cell number 2,000,000 by it with after Accutase enzymic digestion
In PBS, 300g is centrifuged 5min.(2) cell after taking centrifugation abandons supernatant, and 50 microlitres of Geltrex are added in cell precipitation,
It mixes, for use, while the Wnt10b factor is added, making its concentration in Geltrex is 1ug/ml.(3) it will be pregnant 60 days or so
Embryo pig taken out from sow abdominal cavity by laparotomy ventrotomy, but pig fetus is not allowed to be detached from parent, with punch in its back skin
The wound of area about 7mm2 is manufactured on skin.(4) Geltrex for being mixed with skin organ cell that will be prepared in step (2)
It is transplanted to embryo's porcine skin wound surface.(5) embryo pig wound surface is covered with 3M film, then embryo pig is placed and returns to parent
In amnion, suture amnion (refers to Fig. 3).(6) farrowing sow is normally raised, piggy one month or so after normal labor, is cut thin
The skin histology of born of the same parents' transplanting, and after fixed dyeing, hair follicle structure is observed, analysis hair follicle regeneration situation (refers to Fig. 4).
2, Morphological Identification and the cell origin analysis of hair follicle are regenerated
Tissue morphology is carried out to the skin histology of the stem cell transplantation of experimental group and control group in embodiment 3 and embodiment 4
Identification is learned, it is specific as follows: to prepare epidermal stem cells regeneration hair follicle structure organization slice: (1) cutting embodiment 3 and embodiment respectively
The skin tissue area of the stem cell transplantation of experimental group and control group in 4,4% paraformaldehyde is fixed overnight, and PBS impregnates 3 hours,
30% sucrose is dehydrated 8 hours.(2) OCT investing tissue, frozen section take the histotomy of 10 μ m thicks.(3) by histotomy
Naturally dry.(4) histotomy is washed three times, every time 5 minutes with PBS.(5) with 0.2-0.5%tritonX-100, (PBS matches
System) permeable membrane processing 10 minutes is done to histotomy.(6) permeable membrane treated histotomy is washed three times, every time 5 minutes with PBS.
(7) histotomy 2%BSA is closed 30 minutes, is then washed twice with PBS.(8) antibiosis is added in the histotomy after closing
The 1 of object element Cy3 label is anti-, is incubated at room temperature 3 hours.(9) histotomy that connection 1 resists is washed three times, every time 5 minutes with PBS.
(10) 0.5 μ g/ml DAPI (PBS preparation) is added to dye 10 minutes.(11) stained tissue is sliced and is washed three times with PBS, removed
Extra DAPI.(12) 20 μ l mountant mountings are added in the histotomy for removing extra DAPI.(13) the good tissue of mounting
Stained specimens are placed in 4 degree of refrigerators, can be stored 2 weeks or more.The tectology of histotomy is identified: using Laser Scanning Confocal Microscope
Histotomy is observed, it has been observed that there is the formation of hair follicle structure in the regeneration skin histology of embodiment 2, structural form and just
Hair follicle in normal skin is similar；Human archeocyte specific marker MAB1281 coloration result is shown simultaneously, regenerates the cell of hair follicle all
Show that MAB1281 is positive, it was demonstrated that newborn hair follicle be generated from the differentiation of people's multipotent stem cells of transplanting (result is with reference to figure
5)。
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. a kind of method for obtaining artificial hair follicle characterized by comprising
Human pluripotent stem cells are implanted into the skin of mutant embryos, the mutant embryos are in hair formation stages；
There is the embryo of human pluripotent stem cells to continue to cultivate in xenogenesis parent transplanting, until embryo gives birth to；And
Transplanting has the hair follicle in the skin of human pluripotent stem cells in separation embryo, to obtain the artificial hair follicle.
2. the method according to claim 1, wherein the human pluripotent stem cells be induce multi-potent stem cell, institute
Further comprise before stating transplanting: described induce multi-potent stem cell being divided into skin organoid, the skin organoid includes
Ectoderm cell, mesoblastema and neural endoderm cell；
Optionally, described to induce multi-potent stem cell from blood cell or Skin Cell.
3. according to the method described in claim 2, it is characterized in that, described induce multi-potent stem cell is divided into skin organoid
Include:
Described induce multi-potent stem cell is subjected to suspension culture, to obtain 3D cell ball；
The 3D cell ball further progress is broken up and is cultivated, to obtain skin organoid.
4. according to the method described in claim 2, it is characterized in that, described induce multi-potent stem cell is divided into skin organoid
It is to be carried out under conditions of the multipotential stem cell and BMP-4, SB431542, FGF-2 and LND contact；
Preferably, the concentration of the BMP-4 is 50ng/mL.
5. according to the method described in claim 4, it is characterized in that, further comprising carrying out the skin organoid at digestion
Reason, to obtain unicellular mixed liquor；
Optionally, the unicellular mixed liquor is mixed with matrigel or gel-like substance；
Optionally, the matrigel includes being selected from least one of Geltrex and ColI.
6. the method according to claim 1, wherein the human pluripotent stem cells include selected from hair follicle stem cells,
Skin progenitor cell and other stem cells with hair follicle formation or inducibility；
Optionally, the human pluripotent stem cells include the multipotential stem cell of gene modification or induce multi-potent stem cell；
Optionally, the gene modification is gene knockout or gene expression.
7. the method according to claim 1, wherein further comprising before the transplanting: by people's multipotency
Stem cell mixes with the factor for promoting hair follicle to be formed；
Optionally, the factor for promoting hair follicle to be formed is the Wnt10b factor；
Optionally, the concentration of the Wnt10b factor is 1ug/mL；
Optionally, the mutant embryos include being selected from Pig embryos, primate embryonic, rabbit embryonic, ox embryo and sheep embryo
At least one of.
8. the method according to claim 1, wherein the separation is in embryo puerperal 0th~6 month
It carries out.
9. a kind of artificial hair follicle, which is characterized in that obtained by method according to any one of claims 1 to 8.
10. a kind of method of hair follicle plantation, which is characterized in that artificial hair follicle as claimed in claim 9 is inoculated into autologous skin
Surface.
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| CN114317404A (en) * | 2021-12-17 | 2022-04-12 | 上海纳米技术及应用国家工程研究中心有限公司 | Culture medium formula suitable for in-vitro culture of hair follicle stem cells and culture method for in-vitro 3D hair follicle stem cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186969A (en) * | 2008-06-25 | 2011-09-14 | 国家健康与医学研究院 | Methods for preparing human skin substitutes from human pluripotent stem cells |
| US20160213717A1 (en) * | 2015-01-28 | 2016-07-28 | The Trustees Of The University Of Pennsylvania | Compositions and methods for generation of human epithelial stem cells |
| WO2017070506A1 (en) * | 2015-10-21 | 2017-04-27 | Indiana University Research And Technology Corporation | Derivation of human skin organoids from pluripotent stem cells |
| CN106676060A (en) * | 2017-01-09 | 2017-05-17 | 塔里木大学 | Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration |
-
2018
- 2018-01-31 CN CN201810094681.0A patent/CN110093306B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186969A (en) * | 2008-06-25 | 2011-09-14 | 国家健康与医学研究院 | Methods for preparing human skin substitutes from human pluripotent stem cells |
| US20160213717A1 (en) * | 2015-01-28 | 2016-07-28 | The Trustees Of The University Of Pennsylvania | Compositions and methods for generation of human epithelial stem cells |
| WO2017070506A1 (en) * | 2015-10-21 | 2017-04-27 | Indiana University Research And Technology Corporation | Derivation of human skin organoids from pluripotent stem cells |
| CN106676060A (en) * | 2017-01-09 | 2017-05-17 | 塔里木大学 | Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration |
Non-Patent Citations (2)
| Title |
|---|
| 冯万有等: "CRISPR/Cas9***联合人多能干细胞制备嵌合体猪培育人源化器官的研究进展", 《深圳中西医结合杂志》 * |
| 纪影畅等: "大鼠胚胎皮肤毛囊形态发生与Wnt-10b/β-catenin的表达", 《中国美容整形外科杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317404A (en) * | 2021-12-17 | 2022-04-12 | 上海纳米技术及应用国家工程研究中心有限公司 | Culture medium formula suitable for in-vitro culture of hair follicle stem cells and culture method for in-vitro 3D hair follicle stem cells |
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