CN107267448A - A kind of induced fibroblast is the method for in vivo bioreactor - Google Patents
A kind of induced fibroblast is the method for in vivo bioreactor Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/82—Undefined extracts from animals from invertebrates
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
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Abstract
The present invention relates to a kind of method that induced fibroblast is in vivo bioreactor, belong to cell biology and regenerative medicine field.This method is for current by need carrier mediated a kind of with transgeneic procedure, Experimental Establishment and technical merit require cartilage cell security of a relatively high, produce relatively poor the problems such as, invented utilization fish oocyte extract induced fibroblast reprogramming be in vivo bioreactor method of the fibroblast transdifferentiation for generally existing in cartilage cell's method.Because the in vivo bioreactor produced using this method is only obtained by the induction of fish oocyte extract and the directional induction culture of chondroblast inducing culture, it is not related to foreign gene importing, experimental implementation is relatively easy, the in vivo bioreactor security of generation is relatively preferable, is had broad application prospects in cartilage tissue engineered and regenerative medicine field.
Description
Technical field
The present invention relates to a kind of method that induced fibroblast is in vivo bioreactor, belong to cytobiology technology
And regenerative medicine field.
Background technology
Cartilaginous tissue（Cartilage tissue）It is a kind of group rich in materials such as proteoglycan, collagen and hyaluronic acids
Knit, be made up of cartilage cell and cartilage matrix, it is main in vivo to play a part of to support and reduce friction.But cartilaginous tissue lacks blood
Pipe, therefore, it is just difficult to repair that cartilaginous tissue, which once sustains damage, even small cartilage tissue damage, it is also difficult to repair naturally
It is multiple.
At present, the method clinically for cartilage tissue damage reparation mainly has cartilage and subchondral bone removal, micro fractures
With subchondral bone drilling, bone and cartilage autotransplantation and Bone and osterochordral allografts, cartilage or periosteum transplanting etc.（Liu Xiaofang, waits
Chinese orthopaedic trauma magazine, 2014,16 (6):537-539）.These methods to a certain extent can be to cartilage tissue damage
Repaired, but there is distinct disadvantage, such as micro fractures and subchondral bone boring method can make stem cell reach damage location,
But because local environment is different with mechanical stress, stem cell is difficult to Chondrocyte Differentiation；Bone and cartilage autotransplantation safely, effectively,
But new wound can be brought to patient；Allograph bone cartilage transplantation avoids the new wound that bone and cartilage autotransplantation is brought to patient
Wound, but there is donor source limitation, immunological rejection and transmission risk etc..
The treatment for developing into cartilage tissue damage of cartilage tissue engineered technology provides a kind of new thinking and strategy
（Vacanti CA, et al., Plast Reconstr Surg, 1991, 88(5): 753-759; Cao Y, et
al., Transplant Proc, 1994, 26(6): 3390-3392）.In cartilage tissue engineered, seed cell be its most
One of basic key element.Being presently used for cartilage tissue engineered cell mainly has autologous or allograft chondrocytes, genetic modification thin
Born of the same parents, embryonic stem cell, mescenchymal stem cell etc..Immunological rejection is not present in Autologous Chondrocyte transplanting, without induction differentiation
Culture can be used directly, and be optimal cartilage tissue engineered seed cell, but materials are limited, and ability of cell proliferation is low, in vitro
Culture is easy to lose original phenotype（Marlovits S, et al., J Bone Joint Surg, 2004, 86(5):286-
295）；Allogenic chondrocytes are that Autologous Chondrocyte transplanting preferably substitutes cell, it is to avoid Autologous Chondrocyte collection
The new wound caused, but donor source is limited, there is transmission risk；Genetically modified cell can pass through technique for gene engineering
Cytokine gene is imported in cartilage cell, makes it in the cartilaginous lesion site expression cell factor, promotes cartilage damage reparation
（Sellers RS, et al., J Bone Joint Surg, 2002, 79(6):1452-1463）, but channel genes may
There is potential security risk；The differentiation potential of embryonic stem cell is the most comprehensive, but its immunogenicity is also worst, is easily formed in vivo
Teratoma, and it is related to serious dispute of ethic；Though the Tissue distribution of mescenchymal stem cell is relatively broad, its quantitative proportion is relatively low
And differentiation capability is often restricted（Covas D, et al., Brazilian Journal of Medical and
Biological Research, 2003, 36(9): 1179-1183; Kestendjieva S, et al., Cell
biology international, 2008, 32(7): 724-732; Yamazaki H, et al., Stem Cells,
2007, 25(1): 78-87）.
Reprogramming technology and pedigree reprogramming technology（Lineage reprogramming）Develop into cartilage cell's
Source opens new approach.So-called reprogramming technology is that ripe adult cell is first changed into induced multi-potent stem cell
（Induced pluripotent stem cells, iPSCs）, other types of cell is then further divided into, and compose
Be reprogramming technology be then be another type of thin directly by the direct transdifferentiation of adult cell without the multipotential stem cell stage
Born of the same parents.At present, scientist has differentiated bone and cartilaginous tissue using induced multi-potent stem cell（Hong SG, et al., Cell
Reports, 2014 , 7 (4) :1298-1309）, still, reprogramming at present and pedigree reprogram the maximally efficient side of technology
Method is the transcription factor mediated method based on carrier, because this method is related to channel genes, so in the presence of potential potential safety hazard,
Therefore strict limitation is needed in clinical practice.
In summary analyze, cartilage tissue damage reparation still suffers from problems, the particularly seed for injury repair
Cell derived problem, still needs to find that a kind of preparation method is relatively easy, the relatively good adult cell of security is divided into cartilage
The method of cell.Based on this, it is soft using fish oocyte extract induced fibroblast that the present invention, which attempts to establish a kind of,
The method of osteocyte like cell（In view of academic preciseness, the cartilage cell of induction and normal chondrocyte are in epigenetic pattern
Aspect may be incomplete same, therefore, and the cell for inducing Hou Ali Xinlan positive staining is referred to as cartilage cell by patent of the present invention
Like cell）.All steps of the inventive method are not related to channel genes operation, the in vivo bioreactor security of generation compared with
Good, experimental implementation is relatively easy, and the repeatability of experiment is also preferable.
The content of the invention
It is an object of the invention to for being deposited in existing cartilage tissue engineered middle cartilage cell source and method of inducing differentiation
The problem of, invented that a kind of experimental implementation is relatively easy, experimental repeatability relatively preferably, the in vivo bioreactor peace that produces
Full property is relatively good, utilize the method that fish oocyte extract induced fibroblast is in vivo bioreactor.
Induced fibroblast provided by the present invention comprises the following steps for the method for in vivo bioreactor.
（1）The preparation of derivant：Derivant is crucian oocyte extract, and preferably yolk is full of the fish in phase period
Ovum, under sterile and cryogenic conditions, takes out and is ground, filters and determination of protein concentration after fish-egg, prepare for inducing into
The fish oocyte extract of fibrocyte.
（2）It is fibroblastic to prepare：The skin histology of human or animal is fully shredded under aseptic condition, is containing ammonia benzyl
Primary and Secondary Culture is carried out in the DMEM culture mediums of penicillin and streptomysin, the fibroblast for induction is obtained.
（3）Fibroblastic induction：Utilize the step（1）The derivant of middle preparation is to the step（2）It is middle to prepare
Fibroblast induced, make fibroblast epigenetic reprogramming be induced multi-potent stem cell.
（4）The identification of induced multi-potent stem cell：Using immunohistochemical method, according toSox2、NanogWithOct4/3Base
Because expression is identified fibroblastic induction situation.
（5）Induction from induced multi-potent stem cell in vivo bioreactor：According to the step（4）Middle induced multi-potent
The qualification result of stem cell, by the step（3）The induced multi-potent stem cell of middle acquisition is in chondroblast inducing culture
It is oriented induction.
（6）The identification of in vivo bioreactor：The step is identified using A Li Xinlan colouring method and HE colouring methods
（5）Differentiation situation from middle induced multi-potent stem cell in vivo bioreactor.
The step（1）The fish-egg mother cell extract solution storage concentration of middle preparation is 10mg/ml, and condition of storage is 4 DEG C.
The step（2）In be used to cultivate fibroblastic culture medium comprising 100U/ml ampicillins and 100mg/
Ml streptomysin.
The step（3）In be used for fibroblast induce final concentration of 10 ~ 20 μ g/ml of fish oocyte extract,
Fibroblast for induction is the fibroblast of the forth generation human or animal of culture, and fibroblast is in addition fish-egg mother
The Fiber differentiation time in the culture medium of cell extract is 72 hours.
The step（4）The identification of middle induced multi-potent stem cell uses anti-Sox2, Nanog and Oct4/3 antibody of people.
The step（5）Middle chondroblast inducing culture component includes：Fill in DMEM in high glucose basal medium, 10nM
Meter Song（Dex）, 100mg/ml Sodium Pyruvates（Sodium pyruvate）, 10ng/ml TGF-βs 3,50mg/ml ascorbic acid
（Ascorbic acid）, 40mg/ml proline（Proline）With ITS additives；ITS additive components are：Final concentration is distinguished
For 6.25mg/ml bovine insulins（Bovine insulin）, 6.25mg/ml transferrins（Transferrin）、6.25mg/ml
Selenic acid（Selenous acid）, 5.33mg/ml linoleic acid（Linoleic acid）, 1.25mg/ml bovine serum albumin(BSA)s
（Bovine serum albumin）.Fiber differentiation time of the induced multi-potent stem cell in chondroblast inducing culture is
21 days, change a subculture within every 3 days.
The step（6）Middle in vivo bioreactor identifies that the A Li Xinlan dye liquor pH value used is 1.0.
The substantive distinguishing features and marked improvement of the present invention are that the invention provides a kind of acquisition of non-transgenic mediation is soft
The method of osteocyte like cell, the relatively good in vivo bioreactor of security can be obtained using this method.The inventive method
Another distinguishing feature be that the method for the present invention is not related to vector construction and transgeneic procedure, experimental implementation is relatively easy, real
Test equipment and technical requirements are not high, had broad application prospects in cartilage tissue engineered and regenerative medicine field.
Brief description of the drawings
Fig. 1 is the human fibroblasts of culture.
Fig. 2 is Sox2, Nanog and Oct4/3 immunohistochemical staining figure of induced multi-potent stem cell.A figures are Sox2 dyeing
Figure；B figures are Nanog colored graphs；C figures are Oct4/3 colored graphs；D figures are control.
Fig. 3 is after induced multi-potent stem cell is dyed in chondroblast inducing culture after Fiber differentiation through A Li Xinlan
Result figure.
Fig. 4 is the result after induced multi-potent stem cell is dyed in chondroblast inducing culture after Fiber differentiation through HE
Figure.
Embodiment
Technical scheme is further illustrated with reference to the accompanying drawings and examples.Embodiment is only for the purposes of more preferably reason
Solution is of the invention and is not limited to the scope of the invention.
Embodiment 1.
The preparation of derivant.
The present embodiment derivant is used for fibroblastic reprogramming, and derivant is crucian fish oocyte extract, crucian carp
The preferred yolk of fish fish-egg is full of phase period, and its preparation method is.
（1）Crucian is totally submerged and soaked 15~20 minutes in 75% alcohol, fully carries out disinfection, prevents to greatest extent
The only pollution of bacterium etc..
（2）To take out fish-egg under conditions of sterile in fume hood, in the mortar for then inserting precooling, mortar is placed in ice
It is upper to be fully ground.
（3）After the completion of grinding, 1000rpm centrifugation 10min under the conditions of the physiological saline of precooling, 4 DEG C are added, then 4 DEG C of bars
3000rpm centrifuges 10min under part, takes supernatant.
（4）Using 0.22 micron openings filter to the step（3）The supernatant of middle acquisition is filtered, and filtrate is preparation
Fish oocyte extract, the protein concentration of the fish oocyte extract prepared is determined using Coomassie Brilliant Blue.
（5）According to the step（4）The protein concentration for the fish oocyte extract that middle utilization Coomassie Brilliant Blue is measured,
10mg/ml fish oocyte extract storing liquid is diluted to the DMEM/F12 culture mediums of precooling, 4 DEG C save backup.
Embodiment 2.
The preparation of human skin fibroblasts.
The present embodiment fibroblast derives from one full year of life at age 20-25, the prepuce tissues of healthy male, its preparation method bag
Include following steps.
（1）The collection of fell skin tissue：One full year of life at age 20-25, the prepuce tissues of healthy male are selected, aseptically
Clip about 1 × 1cm2It is standby.Prepuce tissues are provided by PLA's Kunming hospital general Urology Surgery, and prepuce tissues are used by understanding
The approval of Fang Jun Kunming hospital general Medical Ethics Committee, the collection and the use equal informed consent of patient of prepuce tissues.
（2）The preparation of human skin fibroblasts：By the step（1）The skin histology of middle collection with physiological saline repeatedly
It is placed in 1 × PBS solution containing 100U/ml ampicillins and 100mg/ml streptomycin sulphates and flushes three times after flushing, cuts
Fat and connective tissue are abandoned, skin histology is then shredded into 0.5 ~ 1mm3The tissue block of size, adds and contains 200U/ml collagens
The DMEM culture mediums of enzyme, 300U/ml hyaluronidases, under the conditions of being placed in 4 DEG C overnight, then by 1:1 ratio, which is added, to be contained
1 × PBS digestive juices of 0.25% pancreatin and 0.02%EDTA, are placed in 37 DEG C, are incubated 20min in 180rpm air table, be incubated
After the completion of add the DMEM/F12 culture medium 10ml containing 10% hyclone, with terminating reaction；Abandoned after 1000rpm centrifugations 10min
Supernatant, collection cell, are cleaned after 2 times with DMEM culture mediums, cell are adjusted with the DMEM/F12 culture mediums containing 10% hyclone
Quantity is every milliliter about 2 × 105Individual cell is inoculated in six well culture plates, and cell is placed in into 37 DEG C, 5%CO2, 90% damp condition
Lower culture, changes a subculture in every 2 days.Pass through the fibroblast after identification, culture and passage and morphological feature such as accompanying drawing 1
Shown, from accompanying drawing 1, by Secondary Culture, fibroblast flocks together, and is arranged in fusiformis.
Embodiment 3.
The fish oocyte extract induction of human skin fibroblasts.
The human skin fibroblasts prepared in the embodiment 2 are induced with the derivant prepared in the embodiment 1, are made
Human skin fibroblasts epigenetic reprogramming under the induction of fish oocyte extract is induced multi-potent stem cell, its method bag
Include following steps.
（1）Fibroblastic induction：When the human skin fibroblasts prepared in the embodiment 2 are passaged to the 4th
When generation and fibroblastic growth to 80% cell fusion, add protein concentration and enter for 10mg/ml fish-egg mother cell extract solution
Row Fiber differentiation 72 hours, it is induced multi-potent stem cell to make fibroblast epigenetic reprogramming, induces the fish-egg mother cell used
Final concentration of 10 ~ the 20ug/ml of extract.
（2）The identification of induced multi-potent stem cell：When the step（1）After the completion of i.e. complete human skin fibroblasts table
Reprogramming is seen, immunohistochemical method is used for identifying reprogrammed cellSox2、NanogWithOct4/3The expression feelings of three genes
Condition.Sox2、NanogWithOct4/3Three expression conditions as shown in Figure 2, as shown in accompanying drawing 2, rearranging by these three genes
Equal positive expression in journey cell, and cellular morphology changes, and the morphological features, cell such as fusiformis, triangle, polygonal is presented
Core is larger, in oblateness.
Described above, human skin fibroblasts present the substantially special of stem cell after being induced through fish oocyte extract
Levy, i.e., human skin fibroblasts are reprogrammed under the induction of fish oocyte extract for induced multi-potent stem cell, and this is thin
Born of the same parents have the potential broken up to other cell types.
Embodiment 4.
Differentiation from induced multi-potent stem cell in vivo bioreactor.
Induced multi-potent stem cell comprises the following steps to the differentiation of in vivo bioreactor.
（1）Directional induction from induced multi-potent stem cell in vivo bioreactor：Induction in the embodiment 3 is produced
Induced multi-potent stem cell 5 × 105It is individual to be placed in six well culture plates, chondroblast inducing culture is added, in 95% humidity, 37
℃、5%CO2Under the conditions of cultivate 21 days, change a subculture within every 3 days, make induced multi-potent stem cell in vivo bioreactor point
Change.Include into chondrocyte induction media components：DMEM in high glucose basal medium, 10nM dexamethasone（Dex）, 100mg/ml acetone
Sour sodium（Sodium pyruvate）, 10ng/ml TGF-βs 3,50mg/ml ascorbic acid（Ascorbic acid）, 40mg/ml dried meat
Propylhomoserin（Proline）With ITS additives；ITS additive components are：Final concentration is respectively 6.25mg/ml bovine insulins（Bovine
insulin）, 6.25mg/ml transferrins（Transferrin）, 6.25mg/ml selenic acids（Selenous acid）、5.33mg/
Ml linoleic acid（Linoleic acid）, 1.25mg/ml bovine serum albumin(BSA)s（Bovine serum albumin）.
（2）The identification of inducing cell：By the step（1）The middle cell induced adds 2ml, 4% paraformaldehyde and fixed
30min, after the completion of fixing, is embedded with paraffin, is dewaxed after the completion of embedding and dewater treatment, then carries out A Li Xinlan
Dyeing and HE dyeing.
A Li Xinlan colouring method：The tissue of FFPE is carried out after dewaxing and dewater treatment, it is 1.0 to add pH value
A Li Xinlan dye liquor, is incubated after 30min and rinses 5min with flowing water, then with being placed in micro- Microscopic observation A Li after distilled water flushing
The staining conditions of Xinlan.
HE colouring methods：Carried out using conventional H E colouring methods, the tissue of FFPE is dewaxed and dewater treatment
Dyed, be placed in using dehydration of alcohol, dimethylbenzene is transparent, after gummy sealing under microscope respectively using h and E dyestuff afterwards
Observe HE staining conditions.
A Li Xinlan and HE coloration results are respectively as shown in accompanying drawing 3 and accompanying drawing 4, and from accompanying drawing 3, inducing cell can be by Ah
Sharp Xinlan dyes blueness, illustrates that inducing cell being capable of secreting acidic mucopolysaccharide, the compositional characteristic with cartilaginous tissue；By accompanying drawing 4
It can be seen that, the visible substantial amounts of in vivo bioreactor of HE coloration results.
It can be illustrated by above example, can be cartilage cell by fibroblast induction using the method for the present invention
Like cell.
Claims (7)
1. a kind of induced fibroblast is the method for in vivo bioreactor, it is characterised in that comprised the following steps：
（1）The preparation of derivant, derivant is fish oocyte extract, and preferably yolk is full of the crucian in phase period, in nothing
Under bacterium and cryogenic conditions, take out fish-egg and be ground, filter, prepare the fish-egg mother cell induced for fibroblast and extract
Thing；
（2）It is fibroblastic to prepare, the skin histology of human or animal is fully shredded under aseptic condition, is containing ammonia benzyl mould
Primary and Secondary Culture is carried out in the DMEM culture mediums of element and streptomysin, the fibroblast for induction is obtained；
（3）Fibroblastic induction, utilizes the step（1）The derivant of middle preparation is to the step（2）Middle preparation into
Fibrocyte is induced, and fibroblast is changed to induced multi-potent stem cell；
（4）The identification of induced multi-potent stem cell, using ImmunohistochemistryMethods Methods, according toSox2、NanogWithOct4/3Gene expression feelings
Condition is identified fibroblastic induction situation；
（5）Induction from induced multi-potent stem cell in vivo bioreactor, by the step（3）The fibroblast of middle induction and
By the step（4）The successful induced multi-potent stem cell of identification induction is induced in cartilage cell's inducing culture；
（6）The identification of in vivo bioreactor, the step is identified using the dyeing of A Li Xinlan and HE colouring methods（5）Middle induction
Differentiation situation from multipotential stem cell in vivo bioreactor.
2. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（1）The fish-egg mother cell extract solution storage concentration of middle preparation is 10mg/ml, and condition of storage is 4 DEG C.
3. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（2）In be used to cultivate chain of fibroblastic culture medium comprising 100U/ml ampicillins and 100mg/ml
Mycin.
4. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（3）Middle final concentration of 10 ~ 20 μ g/ml of the fish oocyte extract used, the fibroblast used is training
Foster forth generation animal fibroblast cell, induction time of the fibroblast in the culture medium of addition fish oocyte extract
For 72 hours.
5. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（4）The identification of middle induced multi-potent stem cell uses anti-Sox2, Nanog and Oct4/3 antibody of people.
6. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（5）Middle cartilage cell's inducing culture component includes：DMEM in high glucose basal medium, 10nM dexamethasone,
100mg/ml Sodium Pyruvates, 10ng/ml TGF-βs 3,50mg/ml ascorbic acid, 40mg/ml proline and ITS additives, ITS
Additive component is：Final concentration be respectively 6.25mg/ml bovine insulins, 6.25mg/ml transferrins, 6.25mg/ml selenic acids,
5.33mg/ml linoleic acid, 1.25mg/ml bovine serum albumin(BSA)s.
7. a kind of method for using a kind of induced fibroblast described in claim 1 in vivo bioreactor, its feature exists
In the step（6）Middle in vivo bioreactor identifies that the A Li Xinlan dye liquor pH value used is 1.0.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384759A (en) * | 2018-01-16 | 2018-08-10 | 深圳市瑞亚再生医学科技有限责任公司 | The method and its application of induced skin fibroblast reprogramming |
| CN109022356A (en) * | 2018-08-30 | 2018-12-18 | 丰泽康生物医药（深圳）有限公司 | A kind of serum free medium improving mesenchymal stem cells into chondrocytes differentiation |
| CN109112102A (en) * | 2018-09-03 | 2019-01-01 | 丰泽康生物医药（深圳）有限公司 | A kind of raising cultural method of the peripheral blood multipotential cell to Chondrocyte Differentiation |
| CN109136178A (en) * | 2018-08-30 | 2019-01-04 | 丰泽康生物医药（深圳）有限公司 | A kind of raising serum free medium of the peripheral blood multipotential cell to Chondrocyte Differentiation |
| CN112877364A (en) * | 2019-11-29 | 2021-06-01 | 中国医学科学院药物研究所 | Reprogramming induction protocol for direct transformation of subchondral bone cells into articular chondrocytes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661010A (en) * | 2004-02-25 | 2005-08-31 | 上海组织工程研究与开发中心 | Method for inducing fibroblast to form cartilage cells |
| CN102925408A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells |
| CN103857797A (en) * | 2011-07-19 | 2014-06-11 | 帷幄生物技术公司 | Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage |
| CN105670989A (en) * | 2014-11-18 | 2016-06-15 | 朱向情 | Kit and method for effectively inducing somatic cell phenotype reprogramming |
-
2017
- 2017-07-28 CN CN201710626304.2A patent/CN107267448A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661010A (en) * | 2004-02-25 | 2005-08-31 | 上海组织工程研究与开发中心 | Method for inducing fibroblast to form cartilage cells |
| CN103857797A (en) * | 2011-07-19 | 2014-06-11 | 帷幄生物技术公司 | Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage |
| CN102925408A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells |
| CN105670989A (en) * | 2014-11-18 | 2016-06-15 | 朱向情 | Kit and method for effectively inducing somatic cell phenotype reprogramming |
Non-Patent Citations (2)
| Title |
|---|
| N. TSUMAKI等: "iPS cell technologies and cartilage regeneration", 《BONE》 * |
| S. G. HONG等: "Path to the clinic: Assessment of iPSC-based cell therapies in vivo in a non-human primate model", 《CELL REP.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384759A (en) * | 2018-01-16 | 2018-08-10 | 深圳市瑞亚再生医学科技有限责任公司 | The method and its application of induced skin fibroblast reprogramming |
| CN109022356A (en) * | 2018-08-30 | 2018-12-18 | 丰泽康生物医药（深圳）有限公司 | A kind of serum free medium improving mesenchymal stem cells into chondrocytes differentiation |
| CN109136178A (en) * | 2018-08-30 | 2019-01-04 | 丰泽康生物医药（深圳）有限公司 | A kind of raising serum free medium of the peripheral blood multipotential cell to Chondrocyte Differentiation |
| CN109112102A (en) * | 2018-09-03 | 2019-01-01 | 丰泽康生物医药（深圳）有限公司 | A kind of raising cultural method of the peripheral blood multipotential cell to Chondrocyte Differentiation |
| CN112877364A (en) * | 2019-11-29 | 2021-06-01 | 中国医学科学院药物研究所 | Reprogramming induction protocol for direct transformation of subchondral bone cells into articular chondrocytes |
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