CN103146644A - Technical method for differentiating mouse hair follicle stem cells to primordial germ cells in vitro - Google Patents
Technical method for differentiating mouse hair follicle stem cells to primordial germ cells in vitro Download PDFInfo
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Abstract
The invention relates to a technical method for differentiating mouse hair follicle stem cells to primordial germ cells in vitro, comprising the following operating steps of: step 1, separating the tentacle hair follicles of a mouse seven days after the birth by virtue of a mechanical method; step 2, culturing the hair follicle stem cells in vitro: washing the hair follicles via DMEM/F-12, placing the washed hair follicles in 0.25% Trypsin-0.04% EDTA (ethylene diamine tetraacetic acid), digesting for 10 minutes at 37 DEG C, slightly blowing and beating, and uniformly mixing, then adding 10% serum to stop the digestion, cleaning in the DMEM/F-12, passing through a 400-mesh cell sieve, and then transferring to hair follicle stem cell culture solution to culture; step 3, culturing the follicle stem cells with two passages in vitro to form an embryoid body EB; step 4, producing mouse embryonic fibroblasts and feeder layer cells; and step 5, culturing the EB, co-culturing with the feeder layer, and differentiating to the primordial germ cells. Via the method disclosed by the invention, the primordial germ cells having similar shapes and specific molecular marker expressions with in-vivo normal primordial germ cells are obtained.
Description
Technical field:
The present invention relates to the technological method of a kind of mouse hair follicles stem cell to the archeocyte differentiation, particularly relate to a kind of by mouse hair follicles stem cell employing vitro culture mode, through forming embryoid body (EB), induced, the l cell of then take induces as feeder layer adopts training method altogether the method that forms archeocyte.The field of biomedicine technology that belongs to reproductive medicine.
Background technology:
Since 2003, a plurality of experimental group were reported them by the mammals stem cell, comprise human stem cells, induce the sexual cell into each etap.Take in the report that male gamete is target, Toyooka in 2003 etc. at first take the Vasa gene as PGC reporting system (reporter system) and BMP4 be inducible factor, mouse stem cells is induced to the cells into PGCs(archeocyte primordial germ); And the PGCs that then will separate produces sperm with being transplanted in testis after testicular cell mixes.2004, Geijsen etc. utilized SSEA1 for the early germ surface markers, added RA (retinoic acids) inducing mouse stem cell, and success obtains in vitro monoploid ， And it is injected in Mouse Eggs, and development of fertilized ova is to blastula stage.The Nayernia of German Research group etc. utilizes RA inducing mouse stem cell equally, and to take Stra8 and Prm1 be reporting system, and the class spermoblast of acquisition can obtain the offspring after being injected into ovum.In addition, the people such as Nayernia also with mouse marrow stem cell in vitro transdifferentiation gone out the cell of archeocyte and spermatogonium sample.2009, the reports such as the Yu of China research group were successfully induced mouse stem cells into sperm.This research it is worth noting first high expression level DAZL gene as the scheme of inducing gamete.Japanese Hayashi in 2011 etc. utilize mouse embryo stem cell, add the inducible factors such as BMP4, isolate PGCs.After being transplanted to testis, obtaining mature sperm, and obtain mouse of future generation.
Comprehensive progress recently, mostly adopt embryonic stem cell (ESC) as starting material, but ESC is in the early stage of mammalian subject growth, and the difficulty of drawing materials, and therefore adopts adult stem cell to replace ESC can deal with problems better to germline.Hair follicle stem cells is the multipotent adult stem cells that a class has self-renewal capacity and multi-lineage potential.
Summary of the invention:
The object of the invention is to overcome the shortcoming of prior art, the technological method of a kind of mouse hair follicles stem cell in vitro to the archeocyte differentiation is provided.The inventive method is separated latter 7 days mouse antenna section hair follicles of birth, the vitro culture hair follicle stem cells, and by going down to posterity, the hair follicle stem cells vitro culture of twice forms embryoid body (EB); Then the l cell (MEF) of take is feeder layer, adopts the mode differentiation and development of cultivating altogether to form the archeocyte like cell, and have with body in similar form and the neural specific gene expression feature of the archeocyte of normal development.
In order to realize the foregoing invention purpose, the inventive method operates in accordance with the following steps: the first step, utilize mechanical process separating mouse antenna section hair follicle: take off from antenna section skin with the operation surgical forceps latter 7 days mouse hair follicles of being born, be transferred to phosphate buffered saline buffer (PBS, in the operation liquid of pH7.0)+10% foetal calf serum (FCS), in operation liquid, clean, and the sebiferous gland that links together of removal and hair follicle, hair muscle, the impurity such as fat, at fresh DMEM/F-12(cell culture fluid Dulbecco's Modified Eagle Medium, F-12 Nutrient Mixture) in, hair follicle is cleaned, second step, the vitro culture hair follicle stem cells: hair follicle is after DMEM/F-12 washes, be placed in 0.25% Trypsin-0.04% EDTA, 37 ℃ digest 10 minutes, piping and druming adds after mixing 10% serum to stop digestion gently, in DMEM/F-12, cleans, and crosses after 400 order cells sieves to proceed to the hair follicle stem cells nutrient solution and cultivate, take and cultivate the same day as zero generation, within every 4 days, go down to posterity once, the 3rd step, the external formation of hair follicle stem cells EB: when hair follicle stem cells is cultivated the s-generation (11-12 days cultivated) 3-4 days the time again, centrifugal collecting cell, abandon supernatant, add 0.25%Trypsin room temperature digestion 5 minutes, then piping and druming, to unicellular, stops digestion gently, proceed to 24 orifice plate suspension culture after Medium 199 washes twice, be placed in the EB substratum and cultivate, the 4th step, the making of l cell (MEF) cultivation and feeder layer cells: female mouse of conceived 13.5 days are killed and take out the tire mouse, taking-up head, four limbs, internal organ, tail are transferred to phosphate buffered saline buffer by trunk and clean, be placed in 0.25% Trypsin-0.04% EDTA, 37 ℃ digest 10 minutes, after piping and druming mixes gently, add 10% foetal calf serum (FCS) to stop, proceed in the inoblast substratum and cultivate, take and cultivate the same day as zero generation, go down to posterity when cell reaches 90% degree of converging, get 1-3 for inoblast, outwell substratum, add the inoblast nutrient solution that contains 10 μ g ametycins, be placed in incubator 1.5-2 hour, taking-up is cleaned with phosphate buffered saline buffer, with 0.25% Trypsin, adherent inoblast is digested, after stopping, the high sugar of DMEM cleans, and proceeds to 24 orifice plate adherent culture, the 5th step, cultivate altogether to archeocyte (PGC) and break up with feeder layer after EB cultivates: with 0.25% Trypsin digestion EB, extremely unicellular with rifle piping and druming in the room temperature digestive process, with the serum termination, digest, collecting cell, centrifugal, abandoning supernatant, add PGC nutrient solution suspension cell, is 5 * 10 by cell dilution to cell concn
4in individual/ml PGCs nutrient solution, add the resuspended cell of 0.5ml in 24 holes that are covered with the mouse embryo fibroblasts feeder layer, culture plate is placed in to 37 ℃, saturated humidity, 5%CO2 and cultivates, nutrient solution comprises: the high sugar of DMEM, 10% foetal calf serum (FCS), 0.23mM Sodium.alpha.-ketopropionate, 0.1mM NEAA (Non-Essential Amino Acids, non-essential amino acid, Hyclone company), the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 20ng/ml Urogastron (EGF), 40ng/ml Prostatropin (bFGF), 40ng/ml stem cell factor (SCF).
The described hair follicle stem cells nutrient solution of the inventive method second step comprises DMEM/F12,2% B-27,20ng/ml Urogastron (EGF), 40ng/ml Prostatropin (bFGF), 1% mycillin.
The described EB substratum of the inventive method the 3rd step comprises Medium 199 (pH 7.0), 3mg/ml bovine serum albumin (BSA), the 1mg/ml Pp63 glycophosphoproteins, 5ul/ml Regular Insulin Transferrins,iron complexes Sodium Selenite (ITS), 0.23mM Sodium.alpha.-ketopropionate, 1ng/ml Urogastron (EGF), 5mIU/ml follitropin (FSH), 3mIU/ml lutropin (LH).
The described inoblast substratum of the inventive method the 4th step comprises: the high sugar of DMEM, 10% foetal calf serum (FCS), 1% Sodium.alpha.-ketopropionate, 1%NEAA, 1% mycillin.
The inventive method successfully utilizes mechanical process to separate latter 7 days mouse antenna section hair follicles of birth, vitro culture hair follicle stem cells (HFSC), utilize the method for the external formation of HFSC EB to be broken up, after differentiation, with l cell, adopt the mode of cultivating altogether to be induced, obtained with body in normally archeocyte (PGC) form and specific molecular marker are expressed similar PGC.
The accompanying drawing explanation:
Fig. 1 is the separation microgram of latter 7 days mouse antenna section hair follicles of birth.
Fig. 2 is mouse hair follicles Stem cells cultured in vitro microgram.
Fig. 3 is the external evoked formation of the hair follicle stem cells EB microgram of twice of going down to posterity.
Fig. 4 is that the l cell feeder layer is made.
Fig. 5 is that hair follicle stem cells is cultivated altogether to each time period microgram of PGC differentiation with MEF after EB induces.
Whether Fig. 6 expresses normal PGC genes involved for the PGC that detects the hair follicle stem cells differentiation.
The PGC that Fig. 7 is the hair follicle stem cells differentiation carries out fluorescent immunohistochemistry.
Embodiment:
Below in conjunction with accompanying drawing and by specific embodiment, the inventive method is further elaborated.
1, the separation of mouse hair follicles
Hair follicle stem cells (HFSC) derives from latter 7 days mouse antenna section skin follicles of birth.With the operation surgical forceps latter 7 days mouse hair follicles of being born, from antenna section skin, take off, see in Fig. 11, be transferred in the operation liquid of phosphate buffered saline buffer (PBS, pH 7.0)+10% foetal calf serum (FCS, Hyclone company), in operation liquid, wash 3 times, and utilize operation surgical forceps to remove the sebiferous gland linked together with hair follicle, hair muscle, the impurity such as fat, see in Fig. 12, in fresh DMEM/F-12 (Hyclone company), hair follicle is washed 3 times.
2, the vitro culture of hair follicle stem cells
Hair follicle is through DMEM/F-12(Hyclone company) wash 3 times after, be placed in 0.25%Trypsin-0.04%EDTA(Hyclone company), 37 ℃ digest 10 minutes, piping and druming adds 10% serum to stop digestion after mixing gently, in DMEM/F-12(Hyclone company) in wash 3 times, proceed to the cultivation of hair follicle stem cells nutrient solution after crossing 400 order cell sieves, the hair follicle stem cells nutrient solution comprises DMEM/F12(Hyclone company); 2%B-27 (Gibco company); 20ng/ml Urogastron (EGF, Sigma company); 40ng/ml Prostatropin (bFGF, Peprotech company); 1% mycillin (Hyclone company), take and cultivate the same day as zero generation, within every 4 days, goes down to posterity once.Figure 2 shows that mouse hair follicles Stem cells cultured in vitro microgram.In Fig. 2,1 was single cell suspension after the cell sieve, cultivate after 1 day and have obvious cell aggregation thing (in Fig. 2 2) to occur, continue to cultivate after 3 days, it is large that clone ball becomes, sharpness of border (in Fig. 2 3), go down to posterity the very smooth compactness in edge of 1 rear clone, the clone is spherical (4-5 in Fig. 2) one by one, and clone's nodule number of the twice rear cell that go down to posterity further increases (in Fig. 2 6).
3, the external formation embryoid body of hair follicle stem cells (EB)
When hair follicle stem cells is cultivated the s-generation (11-12 days cultivated) 3-4 days the time again, the centrifugal 5min collecting cell of 1500rpm, abandon supernatant, add 0.25%Trypsin(Hyclone company) room temperature digestion 5 minutes, then piping and druming is extremely unicellular gently, stop digestion, after washing twice, Medium 199 proceeds to 24 orifice plates (SARSTEDT company) suspension culture, being placed in the EB substratum cultivates, the EB substratum comprises Medium 199 (Gibco company, pH 7.0), 3mg/ml bovine serum albumin (BSA, Sigma company), 1mg/ml Pp63 glycophosphoproteins (Fetuin, Merck company), 5ul/ml ITS (Regular Insulin Transferrins,iron complexes Sodium Selenite Gibco company), 0.23mM Sodium.alpha.-ketopropionate (Hyclone company), 1ng/ml Urogastron (EGF, Sigma company), 5mIU/ml follitropin (FSH, Sigma company), 3mIU/ml lutropin (LH, Sigma company).Figure 3 shows that the external evoked formation of the hair follicle stem cells EB microgram of twice of going down to posterity.In Fig. 31 be by hair follicle stem cells digestion for unicellular, in Fig. 3,2 is to cultivate the EB precursor formed afterwards in 1 day, EB edge clear and become smooth after 2-3 days, be irregularly shaped (3-4 in Fig. 3).
4, the making of l cell (MEF) cultivation and feeder layer cells
Female mouse of conceived 13.5 days are killed and take out the tire mouse, take out head, four limbs, internal organ, tails etc. are transferred to phosphate buffered saline buffer by trunk and clean 3 times, be placed in 0.25% Trypsin-0.04%EDTA(Hyclone company), 37 ℃ digest 10 minutes, after piping and druming mixes gently, add 10% foetal calf serum (FCS, Gibco company) stop, proceed in the inoblast substratum and cultivate, become fibre medium to comprise: the high sugar of DMEM (Hyclone company), 10% foetal calf serum (FCS, Gibco company), 1% Sodium.alpha.-ketopropionate (Hyclone company), 1%NEAA(Hyclone company), 1% mycillin (Hyclone company), take and cultivate the same day as zero generation, go down to posterity when cell reaches 90% degree of converging, get 1-3 for inoblast, outwell substratum, the inoblast nutrient solution that adds 4ml to contain 10 μ g ametycins (Suo Laibao company), be placed in incubator 1.5-2 hour, taking-up is washed 5 times with phosphate buffered saline buffer, with 0.25% Trypsin(Hyclone company) adherent inoblast is digested, after stopping, the high sugar of DMEM is washed 3 times, proceeds to 24 orifice plate adherent culture.In Fig. 4,1 for to proceed to the feeder layer of 24 orifice plates after 12 hours, and cell converges rate and reaches 50% left and right, and in Fig. 4,2 to be expressed as fibrocyte be GFP negative (for the difference of the PGC with the GFP positive is come).
5, after EB cultivates and feeder layer cultivate altogether to archeocyte (PGC) and break up
Use 0.25%Trypsin(Hyclone company) digestion EB, extremely unicellular with rifle piping and druming in the room temperature digestive process, stop digestion, collecting cell, the centrifugal 5min of 1500rpm with 10% foetal calf serum (FCS, Gibco-BRL); Abandoning supernatant, add a small amount of PGC nutrient solution suspension cell, is 5 * 10 by cell dilution to cell concn
4in individual/ml PGCs nutrient solution; Add the resuspended cell of 0.5ml in 24 holes that are covered with the mouse embryo fibroblasts feeder layer, culture plate is placed in to 37 ℃, saturated humidity, 5%CO
2middle cultivation, nutrient solution comprises: the high sugar of DMEM (Hyclone company), 10% foetal calf serum (FCS, Gibco company), 0.23mM Sodium.alpha.-ketopropionate (Hyclone company), 0.1mM NEAA(Hyclone company), the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol (sigma company), 20ng/ml Urogastron (EGF, sigma company), 40ng/ml Prostatropin (bFGF, Peprotech company), 40ng/ml stem cell factor (SCF, sigma company).In Fig. 5, the feeder layer of the 1 unicellular and beneath paving be digested to for EB is cultivated altogether, in Fig. 5,2 is 1 day a lot of cell attachment afterwards, arrive the 5th day that PGC breaks up, occur that diopter is large, the cell of " glittering " (in Fig. 5 3), the number of these cells is along with the increase of incubation time, number get more and more (4-6 in Fig. 5).
6, RT-PCR detects the PGC sexual cell neural specific gene expression of differentiation
Quantitative PCR uses SYBR
premix Ex TaqTM kit(TaKaRa company) and ABI 7300 real-time PCR instrument (Applied Biosystems), adopt β-actin as reference gene.Detect following gene: Dazl, Fragllis, Stella, Figa, Oct3/4, SCP3.Fig. 6 is presented in the PGC atomization, PGC specific gene Dazl, and Blimp1, Stella expresses higher on the 4th day in differentiation, and C-kit expressed higher at the 6th day, and vasa expressed higher at the 2nd day.
System is as follows:
The preparation of Mix:
Each sample needs repetition 6 times in every secondary response, and negative control (with stoning ribonuclease T. water, replacing the cDNA template) is set, to get rid of application of sample error and template, pollute, template relative quantification method is with reference to ABI7300System Software, take action as internal reference, an optional wild-type sample CT value is that vvt, the relative quantification of standard deviation/standard error and genetic expression are calculated in standard value/10.By t-test, check the differential expression in the different sample of goal gene whether to there is statistical significance.
7, the PGC specific gene of cellular immunofluorescence chemical detection differentiation is expressed
Take out the PGC of differentiation, abandon substratum, PBS washes 2 times, adds 4% paraformaldehyde (Suo Laibao company) room temperature to fix 15 minutes.Or PBS washes 3 times, every all over 5 minutes.Use PBST(PBS+0.5%Trition-100) solution changes thoroughly, room temperature 10 minutes.The PBS(phosphate buffered saline buffer, pH 7.0) wash 5 minutes 1 time.Add the PBST(PBS+0.5%Trition-100 containing 10% lowlenthal serum or horse serum (Suo Laibao company)), room temperature sealing 30-60 minute.Abandon confining liquid, add primary antibodie (1:50-1:200, abcam company) to be diluted in confining liquid, 4 ℃ spend the night or 37 ℃ hatch 2 hours.The PBS of 1%BSA washes 3 times, every all over 5 minutes.Add two anti-(1:50-1:200, green the skies company) to be diluted in two anti-diluents, 37 ℃ of lucifuges are placed 1 hour.PBS washes 3 times, every all over 5 minutes.The green skies of the DAPI(company that adds final concentration), room temperature is placed 5 minutes.PBS washes 3 times, every all over 5 minutes.Add 500 μ l PBS or PBS glycerine (1:1) to take pictures or keep in Dark Place under fluorescent microscope.In Fig. 7,1-3 illustrates that the PGC of differentiation is the Vasa positive and, at cytoplasmic expression, in Fig. 7,4-6 illustrates that the PGC of differentiation is the Dazl positive and, at cytoplasmic expression, the PGC that in Fig. 7, the 7-9 explanation is broken up is the OCT-4 positive and expresses at nucleus.
Claims (4)
1. a mouse hair follicles stem cell in vitro is to the technological method of archeocyte differentiation, it is characterized in that operating in accordance with the following steps: the first step, separating mouse antenna section hair follicle: take off from antenna section skin with the operation surgical forceps latter 7 days mouse hair follicles of being born, be transferred in the operation liquid of phosphate buffered saline buffer pH7.0+10% foetal calf serum and clean, and removal and the hair follicle sebiferous gland, hair muscle, the fat that link together, in fresh cell culture fluid DMEM/F-12, hair follicle is cleaned; Second step, the vitro culture hair follicle stem cells: hair follicle is after DMEM/F-12 washes, be placed in 0.25% Trypsin-0.04%EDTA, 37 ℃ digest 10 minutes, piping and druming adds after mixing 10% serum to stop digestion gently, in DMEM/F12, cleans, and crosses after 400 order cells sieves to proceed to the hair follicle stem cells nutrient solution and cultivate, take and cultivate the same day as zero generation, within every 4 days, go down to posterity once; The 3rd step, the external formation embryoid body of hair follicle stem cells EB: when hair follicle stem cells is cultivated the s-generation again 3-4 days the time, centrifugal collecting cell, abandon supernatant, add 0.25% Trypsin room temperature digestion 5 minutes, then piping and druming, to unicellular, stops digestion gently, proceed to 24 orifice plate suspension culture after Medium 199 washes twice, be placed in the EB substratum and cultivate; The 4th step, the making of l cell cultivation and feeder layer cells: female mouse of conceived 13.5 days are killed and take out the tire mouse, taking-up head, four limbs, internal organ, tail are transferred to phosphate buffered saline buffer by trunk and clean, be placed in 0.25% Trypsin-0.04%EDTA, 37 ℃ digest 10 minutes, after piping and druming mixes gently, add 10% foetal calf serum to stop, proceed in the inoblast substratum and cultivate, take and cultivate the same day as zero generation, go down to posterity when cell reaches 90% degree of converging; Get 1-3 for inoblast, outwell substratum, add the inoblast nutrient solution that contains 10 μ g ametycins, be placed in incubator 1.5-2 hour, taking-up is cleaned with phosphate buffered saline buffer, with 0.25%Trypsin, adherent inoblast is digested, after stopping, the high sugar of DMEM cleans, and proceeds to 24 orifice plate adherent culture; The 5th step, cultivate altogether to archeocyte PGC and break up with feeder layer after EB cultivates: with 0.25% Trypsin digestion EB, extremely unicellular with rifle piping and druming in the room temperature digestive process, with the serum termination, digest, collecting cell, centrifugal; Abandoning supernatant, add PGC nutrient solution suspension cell, is 5 * 10 by cell dilution to cell concn
4in individual/ml PGCs nutrient solution; Add the resuspended cell of 0.5ml in 24 holes that are covered with the mouse embryo fibroblasts feeder layer, culture plate is placed in to 37 ℃, saturated humidity, 5%CO
2middle cultivation; Nutrient solution comprises: the high sugar of DMEM, 10% foetal calf serum, 0.23mM Sodium.alpha.-ketopropionate, 0.1mM Non-Essential Amino Acids, the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 20ng/ml Urogastron, the 40ng/ml Prostatropin, the 40ng/ml stem cell factor.
2. a kind of mouse hair follicles stem cell in vitro according to claim 1 is to the technological method of archeocyte differentiation, it is characterized in that the described hair follicle stem cells nutrient solution of second step comprises DMEM/F12,2% B-27, the 20ng/ml Urogastron, the 40ng/ml Prostatropin, 1% mycillin.
3. a kind of mouse hair follicles stem cell in vitro according to claim 1 is to the technological method of archeocyte differentiation, it is characterized in that the described EB substratum of the 3rd step comprises the Medium 199 of pH 7.0, the 3mg/ml bovine serum albumin, the 1mg/ml Pp63 glycophosphoproteins, 5ul/ml Regular Insulin Transferrins,iron complexes Sodium Selenite, 0.23mM Sodium.alpha.-ketopropionate, 1ng/ml Urogastron, the 5mIU/ml follitropin, the 3mIU/ml lutropin.
4. a kind of mouse hair follicles stem cell in vitro according to claim 1 is to the technological method of archeocyte differentiation, it is characterized in that the described inoblast substratum of the 4th step comprises: the high sugar of DMEM, 10% foetal calf serum, 1% Sodium.alpha.-ketopropionate, 1%Non-Essential Amino Acids, 1% mycillin.
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275928A (en) * | 2013-06-18 | 2013-09-04 | 青岛农业大学 | Technical method for differentiating human skin stem cells in vitro into primordial germ cells |
| CN103320383A (en) * | 2013-07-10 | 2013-09-25 | 杭州市萧山区中医院 | Culture medium for culturing rat hair follicle stem cells |
| CN103468745A (en) * | 2013-07-10 | 2013-12-25 | 杭州市萧山区中医院 | Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells |
| CN103468744A (en) * | 2013-07-10 | 2013-12-25 | 杭州市萧山区中医院 | VEGF165 gene modified hair follicle stem cells and preparation method thereof |
| CN103710297A (en) * | 2013-07-10 | 2014-04-09 | 杭州市萧山区中医院 | Separation culture method of rat hair follicle stem cells |
| CN105039246A (en) * | 2015-08-27 | 2015-11-11 | 青岛农业大学 | Primordial germ cell proliferation method capable of promoting mouse skin stem cell differentiation |
| CN105368773A (en) * | 2015-12-23 | 2016-03-02 | 青岛农业大学 | Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells |
| CN106754660A (en) * | 2016-12-11 | 2017-05-31 | 青岛农业大学 | A kind of hair follicle stem cells vitro differentiation is the method for egg mother cell |
| CN106754661A (en) * | 2016-12-11 | 2017-05-31 | 青岛农业大学 | A kind of skin progenitor cell is divided into the technical method of sperm through ectopic development |
| CN106854640A (en) * | 2017-01-16 | 2017-06-16 | 广东万海细胞生物科技有限公司 | A kind of serum free medium of hair follicle stem cells and preparation method thereof |
| KR20180082082A (en) * | 2017-01-10 | 2018-07-18 | 중앙대학교 산학협력단 | Method for germline stem cell from hair follicle cells and use thereof |
| CN108324993A (en) * | 2018-01-15 | 2018-07-27 | 上海神因生物科技有限公司 | A kind of stem cell complex, preparation method and the application of induction hair regeneration |
| CN110564675A (en) * | 2019-09-30 | 2019-12-13 | 广东华夏健康生命科学有限公司 | Separation and extraction method of hair follicle stem cells |
| CN115197898A (en) * | 2022-07-06 | 2022-10-18 | 北京晶莱华科生物技术有限公司 | Preparation method of hair follicle stem cells |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2460985A1 (en) * | 2001-09-20 | 2003-03-27 | Anticancer, Inc. | Nestin-expressing hair follicle stem cells |
| CN1594554A (en) * | 2003-09-12 | 2005-03-16 | 中国医学科学院基础医学研究所 | Rapid adhesion method for separating riched hair follicle stem cell |
| US20100273231A1 (en) * | 2005-09-20 | 2010-10-28 | Andreadis Stelios T | Multipotent mesenchymal stem cells from human hair follicles |
| CN102329779A (en) * | 2005-10-14 | 2012-01-25 | 深圳市北科生物科技有限公司 | Methods for rejuvenating cells into pluripotent stem cells through mrna |
| WO2012142500A2 (en) * | 2011-04-14 | 2012-10-18 | The General Hospital Corporation | Compositions and methods for autologous germline mitochondrial energy transfer |
-
2013
- 2013-02-27 CN CN201310060665.7A patent/CN103146644B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2460985A1 (en) * | 2001-09-20 | 2003-03-27 | Anticancer, Inc. | Nestin-expressing hair follicle stem cells |
| CN1589103A (en) * | 2001-09-20 | 2005-03-02 | 抗癌公司 | Nestin-expressing hair follicle stem cells |
| CN1594554A (en) * | 2003-09-12 | 2005-03-16 | 中国医学科学院基础医学研究所 | Rapid adhesion method for separating riched hair follicle stem cell |
| US20100273231A1 (en) * | 2005-09-20 | 2010-10-28 | Andreadis Stelios T | Multipotent mesenchymal stem cells from human hair follicles |
| CN102329779A (en) * | 2005-10-14 | 2012-01-25 | 深圳市北科生物科技有限公司 | Methods for rejuvenating cells into pluripotent stem cells through mrna |
| WO2012142500A2 (en) * | 2011-04-14 | 2012-10-18 | The General Hospital Corporation | Compositions and methods for autologous germline mitochondrial energy transfer |
Non-Patent Citations (7)
| Title |
|---|
| JONATHAN A. NOWAK ET AL.: "Hair Follicle Stem Cells Are Specified and Function in Early Skin Morphogenesis", 《CELL STEM CELL》 * |
| MARIA P. DE-MIGUEL ET AL.: "Epiblast-derived stem cells in embryonic and adult tissues", 《INT. J. DEV. BIOL.》 * |
| 刘羿羿: "山羊类胚胎干细胞和PGCs的培养与鉴定", 《中国优秀博士论文全文数据库》 * |
| 史明艳等: "不同浓度的IGF1对山羊毛囊干细胞体外培养的影响", 《中国畜牧兽医》 * |
| 吴纲: "毛囊源性干细胞成牙能力的实验研究", 《中国优秀硕士论文全文数据库》 * |
| 李彩霞等: "干细胞向生殖细胞的分化", 《中国组织工程研究与临床康复》 * |
| 谭晓华: "人多潜能毛囊干细胞的分离培养及其向神经元样细胞诱导分化的研究", 《中国优秀硕士论文全文数据库》 * |
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