CN105441381B - Method of the external evoked human adipose mesenchymal stem cells to functional hepatocyte differentiation - Google Patents
Method of the external evoked human adipose mesenchymal stem cells to functional hepatocyte differentiation Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of method that the mescenchymal stem cell that inducing in vitro human is adipose-derived (hADSCs) is broken up to functional liver cell (iHeps).This method includes the steps that are as follows: isolates hADSCs；External evoked culture hADSCs breaks up to iHeps, and induction atomization is divided into 4 stages: pretreatment stage；Entoderm induction period；At liver induction period；The stage of ripeness of liver cell.Use method of inducing differentiation of the invention, it is only necessary to which ten days or so time just generated the liver cell with liver cell form, gene expression and function enough, was the method that shortest non-transgenic method of current used time realizes hepatocyte differentiation.This is beneficial to the application of individuation clinical treatment.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to dry to a kind of mesenchyma that inducing in vitro human is adipose-derived
The method that cell (hADSCs) breaks up to functional liver cell (iHeps).
Background technique
Liver is the critical organ of an adjusting various physiological processes.Liver diseases, such as hepatitis, cirrhosis and burst
Hepatic injury is to lead to dead major reason.Orthotopic liver transplantation (OLT) is the current unique side for treating whole end-stage liver disease
Method, but since the side effects such as immunological rejection and its shortage of liver donor limit the clinical application of liver transfer operation.Hepatocyte transplantation
It is the alternative medicine for treating the whole liver transfer operation of dyshepatia；Although primary hepatocyte can be separated from donor liver
It arrives, but transplanting needs 1~5 × 10 every time9A liver cell, this just needs to obtain a large amount of donor livers or largely can amplification in vitro
Primary hepatocyte.The reasons such as the shortage and primary hepatocyte amplification in vitro difficulty of donor liver to obtain sufficient can be used for
The liver cell of cell therapy is extremely difficult.In order to solve current this awkward situation, there is an urgent need to can quickly generate a large amount of liver cells
New method.
In the past few years, it was found that a variety of liver outer cell mass that can be used for treating liver diseases, wherein latent with Multidirectional Differentiation
It can be exactly a kind of important cell with potential using value with the mescenchymal stem cell (MSCs) of semo-infinite proliferative capacity.Between
Mesenchymal stem cells can be obtained from the Various Tissues such as marrow, fat, Cord blood, amniotic fluid, scalp, placenta.Wherein, adipose-derived
MSCs (ADSCs) be considered as a kind of mescenchymal stem cell for most having application prospect, adipose tissue accounting between 1:
100, between 1:500, are much higher than mesenchymal stem cell proportion in marrow, there is good answer in regenerative medicine
Use prospect.The source ADSCs is sufficient, is easy to get to wound is small, and the adipose tissue for the patient itself that can have drawn from avoids to exempt from this way
Epidemic disease rejection also solves the obstacle of ethics problem etc..Existing research shows ADSCs in vitro and has to induce differentiation into liver thin
The potentiality of born of the same parents.
Current technology requires about 1 month time from ADSCs to liver cell induction differentiation, so that these inductions point
Change method is not particularly suited for practical clinical.And clinical application needs to shorten as far as possible the time of Differentiation Induction in vitro.Therefore,
The new method as mature hepatocytes (iHeps) that in a short time can efficiently induce ADSCs of exploitation is the one of this field
A problem.
Summary of the invention
The present invention claims being existing ADSCs induction the technical issues of solution, as the method for iHeps, time-consuming, low efficiency
Defect.
The technical solution that the present invention solves technical problem is to provide what the new external evoked hADSCs of one kind broke up to iHeps
Method.Method includes the following steps:
A, hADSCs is isolated from the isolated adipose tissue of people；
B, external evoked culture hADSCs breaks up to iHeps, and induction atomization is divided into following 4 stages:
Pretreatment stage: the RPMI-1640 of the ATRA containing 0.5~2 μM is added in the isolated hADSCs of step a
Culture medium or DMEM/F12 culture medium, carry out Fiber differentiation, and induction time is 20~28 hours；
Entoderm induction period: by pretreated hADSCs in IDE1,1~5 μ Μ for being 50~200nM containing concentration
CHIR99021 and 5~20 μM of LY294002 RPMI-1640 culture medium or DMEM/F12 culture medium in carry out induction training
It supports, induction time is 20~28 hours；
At liver induction period: complete entoderm induction after cell containing concentration be 50~200nM IDE1,10~
The RPMI-1640 culture medium of the LDN-193189 of the FGF4 of 30ng/mL, 5~20 μM of LY294002 and 50~200nM or
Fiber differentiation 36~60 hours in DMEM/F12 culture medium；Be subsequently added into containing concentration be 50~200nM IDE1,10~
The RPMI-1640 culture medium or DMEM/ of the fiber mother cell growth factor 4 (FGF4) of 30ng/mL, 5~20 μM of LY294002
Continue Fiber differentiation in F12 culture medium 20~28 hours；
The stage of ripeness of liver cell: the cell after liver induces is completed into the liver for being 100~200ng/mL containing concentration
Fiber mother cell growth factor 4 (FGF4), the 20~40ng/mL oncostatinM of Porcine HGF (HGF), 10~30ng/mL
(OsM), 1.5~3 × 10-5Mol/L dexamethasone (Dex) and ITS premix Williams ' the E culture of general culture additive
The Fiber differentiation in the stage of ripeness is carried out in base, incubation time is 112~144 hours；Obtain the iHeps differentiated by hADSCs
Cell.
Wherein, entoderm induction period described in above method step b is that pretreated hADSCs is being contained 100nM
IDE1 and 3 μ Μ CHIR99021 RPMI-1640 culture medium or DMEM/F12 culture medium in carry out Fiber differentiation, when induction
Between be 24 hours.
It wherein, is that will complete the cell after entoderm induces containing dense at liver induction period described in above method step b
Degree is 50~200nM IDE1,10~30ng/mL fiber mother cell growth factor 4 (FGF4), 5~20 μM of LY294002 and 50
Fiber differentiation 36~60 hours in the RPMI-1640 culture medium DMEM/F12 culture medium of~200nM LDN-193189；Then plus
Entering containing concentration is 50~200nM IDE1,10~30ng/mL fiber mother cell growth factor 4 (FGF4), 5~20 μM
Continue Fiber differentiation 20~28 hours in the RPMI-1640 culture medium or DMEM/F12 culture medium of LY294002.
Wherein, the stage of ripeness of liver cell described in above method step b is to contain the cell after completion liver induction period
There are the hepatocyte growth factor (HGF) of 150ng/mL, the fiber mother cell growth factor 4 (FGF4) of 20ng/mL, 30ng/mL
OncostatinM (OSM), 2 × 10-5The dexamethasone and ITS of mol/L premixes Williams ' the E culture medium of general culture additive
Middle Fiber differentiation, incubation time 114-140 hours.
Wherein, above method step a isolated hADSCs is carried out by step b the method again after cultivating for 3~7 generations
Fiber differentiation.
Wherein, hADSCs described in the above method is inoculated in the coated culture dish of Type I collagen after progress before step b
Continuous Fiber differentiation.
Wherein, hADSCs described in the above method is inoculated in the coated culture dish of Type I collagen, when cell covers with culture dish bottom
The carry out Fiber differentiation of step b is pressed behind portion.
Meanwhile the present invention also provides the iHeps cells for using the above method to be induced by hADSCs.
The present invention has the advantages that using differentiation method of the invention, it is only necessary to which ten days or so time can generate foot
Enough liver cells with special form, gene expression and function are shortest hepatocyte differentiation methods of current used time.This hair
Bright method can obtain made by the liver cell in the source hADSCs the cell of the autologous adipose tissue from patient for treat hepatopathy at
To be possible, so as to avoid the immunological rejection of variant cell.Secondly, the Induction Process of the method for the present invention is quickly high
Effect, this is beneficial to the timely application of clinical treatment.In addition, being made according to the functional analysis of the liver cell after induction differentiation
Resource of the cell for using the method for the present invention induction to obtain as liver cell, it will help the development of hepatocyte transplantation.Therefore, the party
Method is that application of the hADSCs in liver diseases cell therapy has stepped an important step.
Detailed description of the invention
Fig. 1 is the cellular morphology picture after Fiber differentiation.
Fig. 2 is to carry out ELISA and RT-PCR testing result to the cell that induction terminates.
Fig. 3 is the qPCR testing result of hepatocyte-specific gene expression.
Fig. 4 is that the iHeps obtained using induction carries out improvement CCl4The function test result of the acute liver damage of induction.
Specific embodiment
The method of the present invention can specifically be implemented according to the following steps:
A, hADSCs is isolated from the isolated adipose tissue of people；
B, external evoked culture hADSCs breaks up to iHeps, and induction atomization is divided into following 4 stages:
Pretreatment stage: the RPMI-1640 of the ATRA containing 0.5~2 μM is added in the isolated hADSCs of step a
Culture medium or DMEM/F12 culture medium, carry out Fiber differentiation, and induction time is 20~28 hours；
Entoderm induction period: by pretreated hADSCs in IDE1,1~5 μ Μ for being 50~200nM containing concentration
CHIR99021 and 5~20 μM of LY294002 RPMI-1640 culture medium or DMEM/F12 culture medium in carry out induction training
It supports, induction time is 20~28 hours；
At liver induction period: complete entoderm induction after cell containing concentration be 50~200nM IDE1,10~
The RPMI-1640 culture medium of the LDN-193189 of the FGF4 of 30ng/mL, 5~20 μM of LY294002 and 50~200nM or
Fiber differentiation 36~60 hours in DMEM/F12 culture medium；Be subsequently added into containing concentration be 50~200nM IDE1,10~
The RPMI-1640 culture medium or DMEM/ of the fiber mother cell growth factor 4 (FGF4) of 30ng/mL, 5~20 μM of LY294002
Continue Fiber differentiation in F12 culture medium 20~28 hours；
The stage of ripeness of liver cell: the cell after liver induces is completed into the liver for being 100~200ng/mL containing concentration
Fiber mother cell growth factor 4 (FGF4), the 20~40ng/mL oncostatinM of Porcine HGF (HGF), 10~30ng/mL
(OsM), 1.5~3 × 10-5Mol/L dexamethasone (Dex) and ITS premix Williams ' the E culture of general culture additive
The Fiber differentiation in the stage of ripeness is carried out in base, incubation time is 112~144 hours；Obtain the iHeps differentiated by hADSCs
Cell.
Wherein the separation, culture and amplification of hADSCs can carry out according to a conventional method.
As reference, the present invention introduces method in detail below by taking people's intraabdominal adipose tissue as an example:
The separation of step a people's abdominal cavity fat mescenchymal stem cell: caesarean operation takes out people's abdominal subcutaneous adipose tissues,
It is rinsed 3 times in cold D-Hank ' s liquid, removes macroscopic fibre composition and blood vessel, be shredded into 1mm with elbow tweezers3It is left
Right little particle.By adipose tissue granule be transferred to 50mL centrifuge tube and be added 0.1% Type I collagen enzyme (every gram of adipose tissue adds 2-
5mL), 37 DEG C are placed in, oscillation digestion 30min.After the DMEM-LG culture medium containing 10% serum (FBS) of equivalent is added, it will be centrifuged
Pipe turns upside down, and to further speed up tissue block several times discrete for oscillation, and removes fragment of tissue with strainer filtering.Filtered fluid is in 4 DEG C
1500rpm is centrifuged 10 minutes, and repeated centrifugation process simultaneously discards supernatant.Sedimentation cell is resuspended with erythrocyte lysing buffer, room
Temperature stands 10min with splitting erythrocyte.4 DEG C of 1500rpm are centrifuged 10min, abandon after supernatant that (hADSCs is with containing with complete medium
The DMEM-LG culture medium of 15%FBS and 1% penicillin and streptomysin) cell is resuspended in 75cm2In culture dish, and it is placed in 5%
CO2It is cultivated in 37 DEG C of incubators.It after 1 day, is rinsed culture dish 2-3 times with Hank's balanced salt solution, removes non-attached cell, add
Enter complete medium to continue to cultivate.It changes within every 2 days culture solution 1 time, until cell reaches molten with 0.25% pancreatin after 80-90% is merged
Liquid digestion carries out cell passage.
The hADSCs for having cultivated 3-7 generation is inoculated in the coated culture dish of Type I collagen.When cell covers with culture dish bottom
About 10 days laggard behavior phases induced (condition of culture that hADSCs breaks up to liver cell induction is referring to table 1) at liver
Condition of culture of the table 1.ADSC to liver cell induction differentiation
Part additive used in the present invention is described below:
1, IDE1 (being purchased from tocris company, CAS .1160927-48-9), molecular weight: 306.31；
Chemical formula: C15H18N2O5.
Chemical name: 1- [2- [(2-Carboxyphenyl) methylene] hydrazide] heptanoic acid (1-
[2- [(2- carboxyl phenyl) methylene] hydrazides] enanthic acid).
Structural formula:
2, CHIR99021 (being purchased from selleck company, ct99021) molecular weight: 465.34.Chemical formula: C22H18CL2N8；
No. CAS: 252917-06-09；
Chemical name:
6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-
2-ylamino) ethylamino) nicotinonitrile hydrochloride (6 ((2 ((4- (2,4 dichloro benzene base)-
5- (4- methyl-1 hydrogen-imidazoles -2- base) pyrimidine -2-base) amino) ethyl) amino) nicotinic acid nitrile).
Structural formula:
3, LY294002 (being purchased from selleck company, S1105) molecular weight: 307.34.
Chemical formula: C19H17NO3；No. CAS: 154447-36-6；
Chemical name: 2-morpholino-8-phenyl-4H-chromen-4-one (- 4 hydrogen of 2- morpholine -8- phenyl-benzo
Pyrans -4- ketone).
Structural formula:
4, LDN-193189 (being purchased from selleck company, S2618) molecular weight: 406.48
Chemical name: 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)
Quinoline ((4- (6- (4- piperazine -1- base) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline)).
Chemical formula: C25H22N6；No. CAS: 1062368-24-4；
Structural formula:
The ITS that the embodiment of the present invention uses premix general culture additive for Corning Incorporated's product (ITS
Premix Universal Culture Supplement, 20mL, 1/Pack (Product#354350)), when use illustratively
Book requires addition.
By the following examples, the present invention is more specifically described.
Embodiment one is specifically induced into functional iHeps by hADSCs using the method for the present invention
By after 3 generation of hADSCs secondary culture isolated in people's intraabdominal adipose tissue, it is coated to be inoculated in Type I collagen
In culture dish.After hADSCs covers with culture dish bottom, Fiber differentiation is carried out to hADSCs in aforementioned manners, Fig. 1, which is illustrated, to be lured
Terminate the metamorphosis of (D10) cell from starting (D0) to the 10th day is induced to induce during leading, in the method, hADSCs is more
The Ith stage culture medium is changed, cellular morphology becomes short shuttle shape (Fig. 1, D0-D1) from spindle shape after 1 day, then at liver Fiber differentiation
Base culture 3 days.Finally change culture medium into hepatocyte maturation culture medium, cellular morphology becomes having similar to primary hepatocyte
The cube (Fig. 1, D5-D10) of cell tight contact.Culture medium specific formula and incubation time are as follows:
Condition of culture of the hADSCs to liver cell induction differentiation in 2. embodiment one of table
ELISA detection is carried out to the cell that induction terminates, rear iHeps is compared with the hADSCs (Fig. 2) before induction after induction.We
Further analyze the expression of liver cell specific gene, with the hADSCs and liver primary cell (PHH) not induced be respectively yin,
Positive control finds that at liver Induction Process, the expression of specific gene of liver cell gradually raises (Fig. 2), wherein ALB, AAT
It is the specific expressed albumen of liver cell, and is also the embodiment of mature hepatocytes function.And HNF4 α is immature liver cell
Specific proteins, the high expression in liver development, but expressed in mature liver cell not high.Experimental result is shown to be lured by hADSCs
IHeps cells show after leading differentiation has gone out the characteristic feature of mature hepatocytes.These phenomenons illustrate hADSCs cell at
Ripe liver cell stablizes differentiation.These are the result shows that the liver cell in the source hADSCs has successfully been obtained in our this new methods.
The iHeps that test example one, induction obtain has improvement CCl4The in vivo functionality of the acute liver damage of induction is tested
In order to assess whether iHeps has hepatocyte function, we used above-described embodiments 1 to induce obtained iHeps
To CCl4The acute hepatic injury mice model of induction carries out cell transplantation experiment.
1, the iHeps cell for carrying out obtaining after inducing at liver to hADSCs according to same procedure in example one is taken, is added suitable
0.25% pancreatin of equivalent is paved with ware bottom, digests at 37 DEG C 1 minute, and gently patting culture dish makes cell detachment ware bottom and be dispersed into
Individual cells.
Willianm ' the E culture medium that 5mL contains 10%FBS is added and terminates digestion.The mixed liquor of pancreatin and cell is moved on to
15ml centrifuge tube 1000rpm is centrifuged 3 minutes, removes supernatant, and serum-free williams ' E culture medium 5mL is added, and is suspended again thin
Born of the same parents.It is washed 3 times with the method that the centrifugation of serum-free williams ' E culture medium is resuspended repeatedly, last time determines concentration to 2 × 106/100
μL
2, preparing same concentration is 2 × 106The hADSCs and rHeps of/100 μ L
3,20 NPG (sensible company is tieed up in 6 week old, male, Beijing) mouse are divided into four groups, every group 5, are denoted as conduct
The sham-operation group (sham) of blank control, as the hADSCs group of negative control, as the iHeps group of experimental group and as the positive
The rHeps group of control
4, with by CCl4With olive oil at 5% solution, NPG mouse is injected according to 0.3mL/kg CCl4 weight
Cause acute explosive hepatic failure (Fig. 4) after CCl4 solution.After CCl4 is handled 8 hours, back spleen lower end on the left of mouse is cut
Spleen is carefully hauled out half with cotton swab by the osculum of one 5mm long, transplants hADSCs to hADSCs group mice spleen chamber respectively,
IHeps group mouse spleen transplants iHeps, and rHeps group transplants primary hepatocyte (rHeps) cell, and sham-operation group only injects physiology
Salt water (Fig. 4), every group of cell dosage are 2 × 106/ only.After having injected physiological saline or cell, spleen is put back in vivo, with 4
Trumpeter's art linear slit runs mouth, every 30,000 units of Penicillin of mouse and streptomysin intraperitoneal injection jointly.
5, survivorship curve is analyzed: hADSCs group and sham-operation (sham group) group almost all in CCl4 processing 24 hours are dead
Die that (on day 2 dead (Fig. 4 A), rHeps transplantation group significantly improves acute hepatic failure mouse to a mouse of sham-operation group
Survival rate simultaneously extends time-to-live (Fig. 4 A).In iHeps transplantation group, 2 mouse (in total 5) are completely from acute liver damage
Restore (Fig. 4 A)；
6, the grouping and operating procedure of above-mentioned test are repeated.Experimental group and rHeps group are in CCl4(d0) and processing 7 before processing
After it (d7), mouse respectively take three, totally 4 groups, take the mode of blood to collect the blood of these mouse with eyeball, be statically placed in 4 DEG C of mistakes
Night, 1500rpm are centrifuged 10 minutes, take upper serum, are sent to blood biochemistry and are analyzed, analysis shows that, after processing 7 days, paddy third in serum
Transaminase ALT, glutamic-oxalacetic transaminease AST restore normal level, and iHeps group therapeutic effect and the no significant difference of rHeps group
(Fig. 4 B)；
7, liver, production are taken out into the experimental mice execution dissection before CCl4 processing (d0) and after processing 7 days (d7)
HE histotomy.Fabric analysis shows that iHeps significantly improves CCl4The acute liver damage (Fig. 4 C) of induction, CCl4Before processing
(d0) the incomplete feature of cell karyorhexis, dissolution, cellular swelling, cell membrane (at arrow meaning) of damage liver cell is compared
(d7) is effectively relieved after processing.
Claims (8)
1. the method that external evoked hADSCs breaks up to iHeps, feature the following steps are included:
A, hADSCs is isolated from the isolated adipose tissue of people；
B, external evoked culture hADSCs breaks up to iHeps, and induction atomization is divided into following 4 stages:
Pretreatment stage: the RPMI-1640 culture containing 0.5~2 μM of ATRA is added in the isolated hADSCs of step a
Base or DMEM/F12 culture medium, carry out Fiber differentiation, and induction time is 20~28 hours；
Entoderm induction period: being 50~200nM IDE1,1~5 μ Μ containing concentration by pretreated hADSCs
Fiber differentiation is carried out in the RPMI-1640 culture medium or DMEM/F12 culture medium of CHIR99021 and 5~20 μM of LY294002, is lured
Leading the time is 20~28 hours；
At liver induction period: the cell after completing entoderm induction is being 50~200nM IDE1,10~30ng/mL containing concentration
The RPMI-1640 of fiber mother cell growth factor 4 (FGF4), 5~20 μM of LY294002 and 50~200nM LDN-193189 is trained
It supports in base or DMEM/F12 culture medium Fiber differentiation 36~60 hours；Then containing concentration be 50~200nM IDE1,10~
The RPMI-1640 culture medium or DMEM/F12 of 30ng/mL fiber mother cell growth factor 4 (FGF4), 5~20 μM of LY294002
Continue Fiber differentiation in culture medium 20~28 hours；
The stage of ripeness of liver cell: the cell after liver induces is completed into raw for 100~200ng/mL liver cell containing concentration
The long factor (HGF), 10~30ng/mL fiber mother cell growth factor 4 (FGF4), 20~40ng/mL oncostatinM (OsM), 1.5
~3 × 10-5Mol/L dexamethasone (Dex) and ITS premix it is general culture additive Williams ' E culture medium in carry out at
The Fiber differentiation in ripe stage, incubation time are 112~144 hours；Obtain the iHeps cell differentiated by hADSCs.
2. according to the method described in claim 1, it is characterized by: step a state hADSCs, using Type I collagen enzymic digestion
Method is separating obtained from people's isolated adipose tissue, and cellular morphology is long shuttle-type；Pretreatment stage described in step b is hADSCs
It is handled for 24 hours in the RPMI-1640 culture medium containing 1 μ Μ ATRA.
3. according to the method described in claim 1, it is characterized by: entoderm induction period described in step b is after pre-processing
HADSCs carried out in the RPMI-1640 culture medium containing 100nM IDE1,3 μ Μ CHIR99021 and 10 μ Μ LY294002
Fiber differentiation, induction time are 24 hours.
4. according to the method described in claim 1, it is characterized by: described in step b at liver induction period be will complete entoderm
Cell after induction is containing 100nM IDE1,20ng/mL fiber mother cell growth factor 4 (FGF4), 10 μ Μ LY294002
With Fiber differentiation 48 hours in the RPMI-1640 culture medium of 100nM LDN-193189；Then by its containing 100nM IDE1,
20ng/mL fiber mother cell growth factor 4 (FGF4), 10 μ Μ LY294002 RPMI-1640 culture medium in Fiber differentiation 24
Hour.
5. according to the method described in claim 1, it is characterized by: the stage of ripeness of liver cell described in step b is that will complete liver
Cell after induction period is containing 150ng/mL hepatocyte growth factor (HGF), 20ng/mL fiber mother cell growth factor 4
(FGF4), 30ng/mL oncostatinM (OSM), 2 × 10-5Mol/L dexamethasone and ITS premix general culture additive
Fiber differentiation in Williams ' E culture medium, incubation time 120 hours.
6. described in any item methods according to claim 1~5, it is characterised in that: the step a isolated hADSCs training
Fiber differentiation is carried out by step b the method again after supporting 3-7 generation.
7. described in any item methods according to claim 1~5, it is characterised in that: the hADSCs is inoculated in front of step b
Subsequent Fiber differentiation is carried out in the coated culture dish of Type I collagen.
8. according to the method described in claim 7, it is characterized by: the hADSC is inoculated in the coated culture dish of Type I collagen
In, by the carry out Fiber differentiation of step b after cell covers with culture dish bottom.
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Citations (2)
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|---|---|---|---|---|
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| CN105087469A (en) * | 2015-10-09 | 2015-11-25 | 四川大学 | Method for inducing ADSCs (adipose derived stem cells) to differentiate into iHeps in vitro |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104995294A (en) * | 2012-10-19 | 2015-10-21 | 新加坡科技研究局 | Methods of differentiating stem cells into one or more cell lineages |
| CN105087469A (en) * | 2015-10-09 | 2015-11-25 | 四川大学 | Method for inducing ADSCs (adipose derived stem cells) to differentiate into iHeps in vitro |
Non-Patent Citations (4)
| Title |
|---|
| "Rapid and high-efficiency generation of mature functional hepatocyte-like cells from adipose-derived stem cells by a three-step protocol";Fen Xu 等;《Stem Cell Research & Therapy》;20151005;第6卷(第193期);第1-10页 |
| "人脂肪间充质干细胞的分离鉴定及向肝脏样细胞分化的实验研究";张云巍;《中国优秀硕士论文全文数据库（电子期刊）》;20150315(第03期);正文第21-23页 |
| "全反式维甲酸诱导肝前体细胞向肝细胞分化的研究";白光文 等;《重庆医科大学学报》;20121231;第37卷(第3期);摘要，第204页左栏第3段 |
| "视黄酸核受体β介导的ATRA对骨髓间充质干细胞成神经分化促进作用的实验研究";龚敏;《中国博士学位论文全文数据库（电子期刊）》;20121115(第11期);正文第24-25页 |
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