CN105385651B - Inductive pluripotent stem cells Induction of committed differentiation is method and the liver cell of liver cell - Google Patents
Inductive pluripotent stem cells Induction of committed differentiation is method and the liver cell of liver cell Download PDFInfo
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Abstract
The present invention relates to a kind of method that inductive pluripotent stem cells Induction of committed differentiation is liver cell and liver cells.The Induction of committed differentiation that different culture mediums carries out inductive pluripotent stem cells is added by the different phase in Induction of committed differentiation for this method.This method can observe the variation of cellular morphology at any time in operation, it is ensured that induction is normally carried out, and can fast and efficiently induce required hepatic precursor cells.The inductive pluripotent stem cells Induction of committed differentiation is that the method for liver cell uses the cultivating system without feeder cells, only can obtain liver cell by the way that the induced medium of respective stage is added, and after testing, purity is higher.It is the method for liver cell by using above-mentioned inductive pluripotent stem cells Induction of committed differentiation, induction duration is shorter, high-efficient, and obtained Hepatocellular can be stablized, and function is mature, the problem of pollution there is no stroma cell.
Description
Technical field
The present invention relates to cell engineering fields, are liver more particularly, to a kind of inductive pluripotent stem cells Induction of committed differentiation
The method of cell and liver cell.
Background technique
Allosome liver transplant is considered as the most effective means of current treatment End-stage liver disease, and long-term survival rate can after transplanting
Up to 70%-85%.However limited liver donor is far from satisfying clinical demand, it was reported that the patient of liver transfer operation is waited to exist
The waiting period disease death rate is up to 15%.It include at present being made by membrane filtration or resins exchange for the other treatment method of liver failure
External artificial liver, external application be of the same race or the bioartificial liver of xenogenesis liver cell and internal hepatocyte transplantation etc..
1970s are treated based on cell and are attracted extensive attention, and people's fetal-liver treatment has the effect of good clinic
Fruit.Xiang Dedong etc. has chosen 40 Patients with Chronic Severe Hepatitis B, there is different degrees of out of strength, poor, abdominal distension etc. of receiving digestion
Road symptom treats heavy type hepatitis using non-ecology phenomena joint liver fetus cells suspension in 20 patients, there is 8 conditions of patients
Spontaneous recovery, 4 excessive row Liver Transplantation for Treatment of patient's success, improvement survival rate is up to 60%, using simple non-ecology phenomena branch
Holding treatment improvement survival rate is only 45%, and curative effect is not as good as non-ecology phenomena joint liver fetus cells suspension treatment.Zheng Xuanhe etc.
138 patients are treated using plasma exchange joint fetal hepatocyte suspension, clinic, which improves, survives 99, effectively increases heavy type hepatitis
The improvement cure rate of patient, curative effect is better than that fetal hepatocyte suspension or plasma exchange group is applied alone, and plasma exchange group is applied alone and is applied alone
117 patients of replacement therapy of blood plasma, survival rate is applied alone in liver fetus cells suspension group no significant difference, the result and big island a surname's hero etc.
Only 24% equal report result is consistent.But because tire liver source etc. is there are ethics problem, people's fetal-liver treatment has been prohibited.There is research table
It is bright to be transplanted by carrying out MSC (Mesenchymal Stem Cell, mescenchymal stem cell) to chronic liver failure patient, Ke Yigai
Kind liver function and clinical symptoms, and do not occur serious adverse reaction.And MSC does not express MHC- class Ⅱmolecule, and low expression is even
MHC- class Ⅰmolecule is not expressed, there's almost no immune rejection problems.But autologous bone marrow MSC also has certain limitation: such as
It is low that patients Wits difference influences bone marrow collection, autologous patient cell quantity deficiency and proliferation activity, thus limits clinic
Using.
With in November, 2007, the Liang Ge research group of the U.S. and Japan almost announces human skin cell successfully simultaneously
It is transformed into the inductive pluripotent stem cells that can almost compare favourably with embryonic stem cell --- " iPS cell " has been avoided always for a long time
Dispute of ethic, solve stem cell transplantation immune rejection problems, the advantage of life science basic research and medical domain
Become clear day by day.
Summary of the invention
Based on this, it is necessary to it is thin for the method for liver cell and liver to provide a kind of inductive pluripotent stem cells Induction of committed differentiation
Born of the same parents.
A kind of inductive pluripotent stem cells Induction of committed differentiation is the method for liver cell, is included the following steps:
Step S1 provides or prepares inductive pluripotent stem cells；
Step S2, by the inductive pluripotent stem cells initial incubation in the RPMI- for being added with human activin A and Wnt3a
In 1640 culture mediums, and RPMI-1640 culture medium of the replacement added with human activin A and fetal calf serum during the cultivation process, make
The inductive pluripotent stem cells are induced to limited entoderm to be broken up；
Step S3, the cell initial incubation that step S2 is cultivated are being added with keratinocyte growth factor and tire ox blood
In clear KO/DMEM culture medium, and incubation replacement added with serum substitute, L-Glutamine, nonessential amino acid,
The KO/DMEM culture medium of beta -mercaptoethanol and dimethyl sulfoxide induces the cell of culture to liver system cell directional and breaks up；
Step S4, the cell culture that step S3 is cultivated are being added with fetal calf serum, hepatocyte growth factor, tumor suppression
In the L-15 culture medium of element, dexamethasone and L-Glutamine, promote hepatocyte maturation.
In one of the embodiments, in the step S1, imported using virus infection, plasmid transfection, mRNA,
MiRNA is imported or chemical molecular addition manner imports inducible factor in human cell, is induced the human cell to be divided into and is lured
The property led multipotential stem cell；The inducible factor includes OCT4, SOX2, NANOG and Lin28.
In one of the embodiments, in the step S2, using described added with human activin A and Wnt3a
After RPMI-1640 culture medium culture 22-50 hours, replacement uses the RPMI- added with human activin A and fetal calf serum
1640 culture medium cultures 46-98 hours, and one subculture of every 22-26 hours replacement during follow-up cultivation.
In one of the embodiments, described in the RPMI-1640 culture medium added with human activin A and Wnt3a
The concentration of human activin A is 5-300ng/ml, and the concentration of the Wnt3a is 20-100ng/ml.
Institute in the RPMI-1640 culture medium added with human activin A and fetal calf serum in one of the embodiments,
The concentration for stating human activin A is 10-200ng/ml, and the volumetric concentration of the fetal calf serum is 0.2-2%.
In one of the embodiments, in the step S3, keratinocyte growth factor and tire are added with using described
Behind KO/DMEM culture medium culture 22-98 hours of cow's serum, reuse added with serum substitute, L-Glutamine, nonessential
The KO/DMEM culture medium culture of amino acid, beta -mercaptoethanol and dimethyl sulfoxide 118-170 hours, and during the cultivation process,
Primary corresponding culture medium was replaced every 22-26 hours.
The KO/DMEM culture added with keratinocyte growth factor and fetal calf serum in one of the embodiments,
The concentration of keratinocyte growth factor described in base is 15-100ng/ml, and the volumetric concentration of the fetal calf serum is 2-3%.
It is described in one of the embodiments, to be added with serum substitute, L-Glutamine, nonessential amino acid, β-mercapto
The volumetric concentration of serum substitute described in the KO/DMEM culture medium of base ethyl alcohol and dimethyl sulfoxide is 20%, the L- paddy ammonia
The concentration of amide is 1-3mM, and the mass concentration of the nonessential amino acid is 0.1-15%, and the concentration of the beta -mercaptoethanol is
0.1-0.12mM, the volumetric concentration of the dimethyl sulfoxide are 0.2-2%.
In one of the embodiments, in the step S4, cultivation cycle is 142-242 hours, and each 22-26 is small
Mono- subculture of Shi Genghuan.
It is described in one of the embodiments, to be added with fetal calf serum, hepatocyte growth factor, tumour inhibitor, dexamethasone
And the volumetric concentration of fetal calf serum described in the L-15 culture medium of L-Glutamine be 10%, the hepatocyte growth factor it is dense
Degree is 10-300ng/ml, and the concentration of the tumour inhibitor is 1-150ng/ml, and the concentration of the dexamethasone is 0.05-1.2 μM,
The concentration of the L-Glutamine is 2-3mM.
The present invention also provides a kind of using inductive pluripotent stem cells directional induction described in any of the above-described embodiment point
The liver cell that the method for turning to liver cell constructs.
The present invention is by reasonably using Porcine HGF to provide a kind of inductive pluripotent stem cells of optimization in vitro
Under the conditions of Induction of committed differentiation be hepatic precursor cells method, this method can observe cellular morphology at any time in operation
Variation, it is ensured that induction is normally carried out, and can be more quick and efficiently induce required hepatic precursor cells.The induction
Property multipotential stem cell Induction of committed differentiation be liver cell method using the cultivating system of no feeder (feeder cells), only lead to
The induced medium for crossing addition respective stage can obtain liver cell, and after testing, purity is higher.By using above-mentioned induction
Property multipotential stem cell Induction of committed differentiation be liver cell method, induction duration is shorter, high-efficient, and obtained Hepatocellular energy
The problem of stablizing, being polluted there is no stroma cell.
Detailed description of the invention
Fig. 1 is that cellular morphology changes schematic diagram during hiPS induced synthesis in the embodiment of the present invention, wherein A: Human embryo
Fibroblast, B: the 4th day cellular morphology after virus infection, C: the non-ES sample clone that infection is formed for 2 weeks or so, D-F: people
The typical iPS of the upper secondary culture of feeder is cloned, the clone of the periphery F:iPSCs Spontaneous Differentiation, A-D and F scale is 200 μm, E
Scale is 50 μm；
Fig. 2 is the Almightiness type related gene expression detection schematic diagram in 1hiPSCs of the embodiment of the present invention；
Fig. 3 is in the EB difference divergaence time point in hiPSCs of the embodiment of the present invention and the source hESCs, domestic and abroad triploblastica generation
Table gene expression schematic diagram, wherein SOX17: entoderm, RUNX1: mesoderm, PAX6: ectoderm；
Fig. 4 is the horizontal detection schematic diagram of hiPSCs of embodiment of the present invention telomerase；
Fig. 5 is the immunohistochemical analysis schematic diagram of hiPSCs of the embodiment of the present invention, wherein A:AKP dyeing, B:SSEA-3,
C:SSEA-4, D:TRA-1-60, E:TRA-1-81, F:OCT4, A scale are 200 μm, and B-F scale is 100 μm；
Fig. 6 is that the external triploblastica of hiPSCs of the embodiment of the present invention breaks up detection schematic diagram, wherein A:B-tubulin/
DAPI, B:SMA/DAPI, C:AFP/DAPI, teratoma histotomy show the cell D: cartilage (mesoderm) in triploblastica source；
E: glandular epithelium (entoderm)；F: chromatophore (ectoderm)；
Fig. 7 is the metamorphosis situation of each stage IPS cell of the embodiment of the present invention；
Fig. 8 is immunofluorescence dyeing of embodiment of the present invention AFP, ALB figure；
Fig. 9 is the testing result of RT-QPCR of the embodiment of the present invention
Figure 10 is that ICG of embodiment of the present invention endocytosis and exocytosis are tested；
Figure 11 is that the liver cell in the source iPS of the embodiment of the present invention has the ability of glycogen biosynthesis；
Figure 12 is that the hepatic precursor cells in the source iPS of the embodiment of the present invention have intake low-density lipoprotein ability.
Specific embodiment
It is to inductive pluripotent stem cells Induction of committed differentiation of the invention mainly in combination with drawings and the specific embodiments below
The method of liver cell is described in further detail.
The inductive pluripotent stem cells Induction of committed differentiation of one embodiment is the method for liver cell, is included the following steps:
Step S110 provides or prepares inductive pluripotent stem cells.
In the present embodiment, inductive pluripotent stem cells preferably use virus infection, plasmid transfection, mRNA import,
MiRNA is imported or chemical molecular addition manner imports inducible factor in human cell, such as is directed into fibroblast, is induced
Human cell is divided into inductive pluripotent stem cells.Wherein, inducible factor includes OCT4, SOX2, NANOG and Lin28.At fiber
Cell can be in vitro adult, baby or skin fibroblasts of fetus etc..It is understood that in other embodiments, luring
The property led multipotential stem cell is not limited to break up to obtain by human dermal fibroblasts, can also be using other human cell's buildings
It obtains.
Step S120, by inductive pluripotent stem cells initial incubation in the RPMI- for being added with human activin A and Wnt3a
In 1640 culture mediums, and RPMI-1640 culture medium of the replacement added with human activin A and fetal calf serum during the cultivation process, make
Inductive pluripotent stem cells are induced to limited entoderm to be broken up.
In the step s 120, using the RPMI-1640 culture medium added with human activin A and Wnt3a (i.e. in RPMI-
Human activin A and Wnt3a are added on the basis of 1640 basal mediums, below similarly) after culture 22-50 hours, replacement uses
RPMI-1640 culture medium culture 22-50 hours added with human activin A and fetal calf serum, and during follow-up cultivation
One subculture of every 22-50 hours replacement.In the present embodiment, the RPMI-1640 training added with human activin A and Wnt3a
The concentration for supporting human activin A in base is 5-300ng/ml, and the concentration of preferably 100ng/ml, Wnt3a are 20-100ng/ml, preferably
25ng/ml.The concentration of human activin A is 10- in RPMI-1640 culture medium added with human activin A and fetal calf serum
200ng/ml, the volumetric concentration of fetal calf serum are 0.2-2%.
It further, in the present embodiment, further include dry to inductive pluripotent before inductive pluripotent stem cells culture
Cell is recovered and is proliferated the step of expanding culture.Wherein, the step of recovery includes: by inductive pluripotent stem cells from liquid nitrogen
It is taken out in tank, immediately the constant-temperature thawing in 37 DEG C of constant water bath box, centrifugation abandons supernatant and takes out dimethyl sulfoxide, and ES culture medium is added
Cell precipitation is resuspended, is planted on ready mouse embryo fibroblast feeder cells, changes the liquid once, pass weekly daily
In generation, is primary.It includes: to choose luring of growing fine after detection is determined without mycoplasma, germ contamination that proliferation, which expands the step of culture,
The property led multipotential stem cell clone, hand cut are passaged in the culture dish for being coated with Matrigel glue in advance and cultivate, and train in mTESR
After cultivating cell about 2-4 days in the system of supporting, induced medium is replaced after using the culture of mouse fibroblast cell conditioned medium instead one day
(being added with the RPMI-1640 culture medium of human activin A and Wnt3a).
Step S130, the cell initial incubation that step S2 is cultivated are being added with keratinocyte growth factor and tire ox
In the KO/DMEM culture medium of serum, and serum substitute, L-Glutamine, non-essential amino are added in incubation replacement
The KO/DMEM culture medium of acid, beta -mercaptoethanol and dimethyl sulfoxide induces the cell of culture to liver system cell directional and breaks up；
In step s 130, using the KO/DMEM culture medium culture for being added with keratinocyte growth factor and fetal calf serum
After 22-98 hours, reuse added with serum substitute, L-Glutamine, nonessential amino acid, beta -mercaptoethanol and dimethyl
The KO/DMEM culture medium culture of sulfoxide 118-170 hours, and during the cultivation process, it is primary corresponding every replacement in 22-26 hours
Culture medium.Further, in the present embodiment, it is cultivated added with the KO/DMEM of keratinocyte growth factor and fetal calf serum
The concentration of keratinocyte growth factor is 15-100ng/ml in base, and the volumetric concentration of fetal calf serum is 2-3%.Added with serum
Substitute, L-Glutamine, nonessential amino acid, beta -mercaptoethanol and dimethyl sulfoxide KO/DMEM culture medium in serum replace
Volumetric concentration for object is 20%, and the concentration of L-Glutamine is 1-3mM, and the mass concentration of nonessential amino acid is 0.1-
15%, the concentration of beta -mercaptoethanol is 0.1-0.12mM, and the volumetric concentration of dimethyl sulfoxide is 0.2-2%.
Step S140, the cell culture that step S3 is cultivated are being added with fetal calf serum, hepatocyte growth factor, suppression
In the L-15 culture medium of tumor element, dexamethasone and L-Glutamine, promote hepatocyte maturation.
In step S140, cultivation cycle is 142-242 hours, and one subculture of each 22-26 hours replacement.Into one
Step, in the present embodiment, added with fetal calf serum, hepatocyte growth factor, tumour inhibitor, dexamethasone and L-Glutamine
The volumetric concentration of fetal calf serum is 10% in L-15 culture medium, and the concentration of hepatocyte growth factor is 10-300ng/ml, tumour inhibitor
Concentration be 1-150ng/ml, the concentration of dexamethasone is 0.05-1.2 μM, and the concentration of L-Glutamine is 2-3mM.
Present embodiment is existed by the inductive pluripotent stem cells for reasonably Porcine HGF being used to provide a kind of optimization
Induction of committed differentiation is the method for hepatic precursor cells under conditions in vitro, and this method can observe cell at any time in operation
The variation of form, it is ensured that induction is normally carried out, and rejecting abnormalities can be cloned at any time, so as to more quick and efficiently lure
Hepatic precursor cells needed for export.The inductive pluripotent stem cells Induction of committed differentiation is that the method for liver cell uses nothing
The cultivating system of feeder (feeder cells) only can obtain liver cell by the way that the induced medium of respective stage is added, and
After testing, purity is higher.It is the method for liver cell, induction by using above-mentioned inductive pluripotent stem cells Induction of committed differentiation
The problem of period is shorter, high-efficient, and obtained Hepatocellular can be stablized, and there is no stroma cell pollutions.
The following are specific embodiment parts:
One, used reagent and its equipment
1.iPSC (inductive pluripotent stem cells): it is the mankind that selection human stem cells National Engineering Research Centre, which builds be 2,
It induces multi-potent stem cell.
2. cell culture reagent:
Human activin A (R&D, 338-AC-050, i.e. R&DSystems company, article No. 338-AC-050, below similarly),
Wnt3a (R&D, 1324-WN-010), fetal calf serum (Life, 10099141), keratinocyte growth factor (KGF) (R&D, 251-
KG-050), serum substitute (Life, N10828-028), L-Glutamine (Gibco, 25030-081), nonessential amino acid
(Gibco, 1140-050), beta -mercaptoethanol (Gibco, 21985023), dimethyl sulfoxide (DMSO) (Sigma, D2650), liver
Porcine HGF (HGF) (R&D, 294-HG-025), tumour inhibitor (OSM) (R&D, 295-OM-050), dexamethasone
(Calbiochem, 265005), mTESR (Stem cell, 05850), RPMI-1640 (Life, 11875-085), KO/DMEM
(Life, 10829-018), L-15 (Life, 21083-027), Matrigel glue (BD, 354234).
Two, specific induction and its qualification process
(1) building for people iPSC cell line is
Experimental subjects: the skin fibroblasts of 8-10 weeks isolated aborted fetus, the laboratory sample is from Hunan
Refined hospital, the research of the present embodiment have passed through the middle letter refined reproduction in Hunan and have passed through discussion with heredity Ethics Committee, section hospital, and
And achieve the agreement of aborted fetus relatives.It is understood that in other embodiments, the skin fibroblasts or other mankind
Cell sample can also be derived from some samples and freeze library or treatment mechanism etc..
Tetra- kinds of inducible factors of OCT4, SOX2, NANOG and Lin28 are imported into fiber finer using the method for slow-virus infection
Born of the same parents obtain inductive pluripotent cell (iPSCs).Detailed process is as follows:
By tetra- slow virus plasmids of pOCT4, pSOX2, pNANOG, pLin28, (plasmid is by the high-new Life Science of Hunan Guang Xiu
Co., Ltd's clone's preparation) it is mixed respectively with slow virus packaging plasmid pCMV8.91, pCMV-VSVG, the 293T in culture is added
In cell, cell conditioned medium is collected after 48 hours, contains the lentiviral particle that can be used for infecting in supernatant.
As shown in Figure 1, by above-mentioned 4 kinds of lentiviral particles according to 1:1:1:1 ratio mix after be added culture in skin at
In fibrocyte (Figure 1A), about there is small clone to occur after two weeks, part colony morphology is different from hES clone (Fig. 1 C), part
Typical hES sample is presented in clone, and passage amplification can maintain the form (Fig. 1 D-F) of hES cloning.From 1 × 105HEF in about
Have and is greater than 30 typical case's hES sample clonal growths.The formation efficiency of repeated experiment detection iPS is about 10-5--4.On people feeder
The clone of the hES sample of culture, cell tight, nucleocytoplasmic ratio is big, similar to hES (Fig. 1 D-F).It can be observed simultaneously in incubation
There is Spontaneous Differentiation (Fig. 1 F) in iPS clone periphery.These clones are similar to hES, as hiPS.It is divided into from this plant of fibroblast
From 16 iPS clones, careful detection has been carried out to wherein hiPS-#2 and hiPS-#5.It detects and hESC
The identical clone of characteristic is hiPSC, can be used for testing in next step.Specific detection is as follows:
1.1 the detection of versatility related gene and embryoid body (EB) differentiation gene
The cell of undifferentiated hESCs, iPSCs and EB differentiation different time points is collected, extract RNA carries out totipotency dependency basis
The detection of cause or differentiation gene.Method is as follows:
1) RNA is extracted:
Corresponding cell is collected, the TRIZOL of 1ml, lytic cell are added.Every 1ml TRIZOL adds 0.2ml chloroform, in room temperature
Lower incubation 2-3 minutes.12 at 4 DEG C, the centrifugal force high speed refrigerated centrifuge of 000 × g 15 minutes.Mixture is divided into three after centrifugation
Layer, RNA are present in water sample layer.Water sample layer is transferred in a clean test tube.0.5ml isopropanol is added to be sufficiently mixed.
Mixed sample is incubated at room temperature 10 minutes to and is no more than at 4 DEG C the centrifugal force high speed refrigerated centrifuge 10 of 12,000g
Minute.Remove upper layer suspension.With the ethanol washing RNA precipitate of 75v/v% (i.e. the volume fraction of ethyl alcohol is 75%, below similarly)
Once, it is no more than at 2-8 DEG C centrifugal force high speed refrigerated centrifuge 5 minutes of 7,500g.(air is dry for simple dry RNA precipitate
Dry 5-10 minutes).It is dissolved with the DEPC water of no RNA enzyme, is completely dissolved rear UV spectrophotometer measuring RNA concentration.
2) reverse transcription (RT) synthesizes cDNA:
Using A RevertAidTM first strand cDNA synthesis kit(Fermentas Life
Sciences), carried out referring to specification, the specific steps are as follows: take 1 μ g total serum IgE, 1 μ l of oligo (d T) primer is added
RNase-free water makes 12 μ l of end reaction volume in EP pipe, 70 DEG C of denaturation 5min, cooling rapidly on ice.Then 5X is sequentially added
4 μ l, 10mM dNTP of reverse transcription reaction buffer 2 μ l, 1 μ l of RNase inhibitor are mixed gently, rapid centrifugation, 25 DEG C of processing
5min adds 1 μ l of AMV reverse transcriptase, end reaction volume 20 μ l, 25 DEG C of 10min；42 DEG C of 60min, 70 DEG C of 10min, 4 DEG C of coolings.Institute
It obtains cDNA and places -20 DEG C of preservations or after the PCR amplification target gene that continues.
Using RNA as template, common β-actin primer carries out PCR amplification, determines the pollution without gDNA.
Again using cDNA as template carry out RT-PCR, with detect wherein versatility related gene OCT3/4, NANOG, REX-1,
The expression of SOX2, THY1, TDGF1, TERF1, LEFTB, DPPA2 and FGF4 or triploblastica differentiation represent gene (ectoderm:
Pax6, mesoderm: Runx1, entoderm: Sox17), β-actin is reaction system positive control.PCR reaction condition: 95 DEG C 1 point
30 seconds；30 circulations (94 DEG C 40 seconds, 54 DEG C -64 DEG C 40 seconds, 72 DEG C 40 seconds)；72 DEG C extend 7 minutes.
As shown in Figure 2 A, many undifferentiated ES cell marking genes of RT-PCR display hiPS cell expression, such as OCT3/4,
SOX2, NANOG, reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4),
Developmental pluripotency-associated 2 (DPPA2), and telomerase reverse
Transcriptase (hTERT) etc., it is similar to hES expression.
As shown in Figure 2 B, it is shown in hiPS cell using the primer detection for the expression of interior external source OCT4, NANOG in existing
The expression of OCT4, NANOG of source property, it is seen that total OCT4 in hiPS, the expression of NANOG are suitable with endogenous expression.
As shown in figure 3, the external Spontaneous Differentiation experiment display of hiPSC, as hESC, the external Spontaneous Differentiation of hiPSC is formed
EB have in, in, the representative gene expression of outer three germinal layers, illustrate that hiPSC has versatility as hESC.
1.2 Analysis of Telomerase Activity
Undifferentiated iPSCs is collected, using TRAPeze Telomerase detection kit (Chemicon) kit,
According to illustrate carry out Telomerase activity.HERT is highly expressed in multipotential cell, as shown in figure 4, the present embodiment detection is also sent out
Show people hiPS also high expression telomerase activation.
1.3AKP dyeing and immunofluorescence dyeing
1) AKP is dyed:
Using Fast Red Substrate Pack (Zymed Laboratories) kit and AKP kit
(invitrogen) AKP dyeing is carried out according to corresponding specification.
(1) hESCs of culture is removed into residual media as far as possible；
(2) PBS is added to wash twice, removes PBS；
(3) the fixed hESCs clone of 4% paraformaldehyde of 4 DEG C of pre-coolings is added, sets the fixed 15-20min of room temperature；
(4) fixed cell is taken out, is washed 2 times using distilled water；
(5) illustrate according to kit, dye liquor dyeing is added in the dye liquor dyeing of fresh configuration；
(6) ordinary stain 10 minutes at room temperature；
(7) it when dyeing is satisfied with, is washed with distilled water 3 times；
(8) it microscopically observation and images.
2) immunofluorescence dyeing
The fixed undifferentiated iPS clone of paraformaldehyde, makees the dyeing of multipotent stem cells antigen, comprising: OCT4, SSEA-3,
SSEA-4 and TRA-1-60, TRA-1-81；Cell after the fixed EB of paraformaldehyde is adherent carries out the dyeing of triploblastica specific marker,
It include: AFP (entoderm), β-tubulin (ectoderm), SMA (mesoderm).The specific method is as follows:
Immunofluorescence dyeing step:
(1) plus the paraformaldehyde solution of 4wt% (i.e. mass concentration, below similarly) fixed test cell 15 divides at room temperature
Clock adds PBS to clean 3 times；
(2) in intracellular and core dyeing with 0.1% triton-X-100 permeable membrane 10min (membrane antigens dyeing omission this
Step)；
(3) add confining liquid i.e. 10% donkey or lowlenthal serum processing (consistent with secondary antibody source) 45 minutes, PBS is added to clean 3 times；
(4) it is proportionally added into corresponding primary antibody, 4 DEG C of overnight incubations add PBS to clean 3 times；
(5) plus fluorescence secondary antibody room temperature is protected from light incubation 1 hour, is cleaned 3 times with PBS；
(6) plus DAPI contaminates core, and room temperature is protected from light incubation 5 minutes, is cleaned 2 times with PBS；
(7) fluorescence microscopy microscopic observation result.
Vitro differentiation ability in 1.4hiPSCs body
For the vitro differentiation ability for detecting hiPS, the present embodiment forms EB using Maitland culture.By iPS grams under body formula mirror
It is grand to be cut to suitable size (more slightly larger than passage clone's agglomerate), it will gently be cloned under agglomerate shovel with TIP, cloning block will be shaken to training
Ware center is supported, is transferred in the Micro-Organism Culture Dish (petridish) added with the 60mm of 4ml EB culture medium, 37 DEG C, 5%CO2Training
It supports and is cultivated in case.Liquid is changed every other day.The dead cell on the surface EB is removed, by EB group to EB gently pressure-vaccum with the tip head of 1ml when changing liquid
Block shakes to ware center, is transferred in the Micro-Organism Culture Dish (Petri dish) added with fresh EB culture medium.After suspending culture 10 days,
EBs is transferred in coated 24 orifice plate of FBS.As shown in figure 5, being exempted from after being continued adhere-wall culture 1 week with identical culture medium
The dyeing of epidemic disease cell fluorescence, Immuncytochemical detection find β-tubulin (ectoderm), SMA (mesoderm), AFP (entoderm)
It is positive.For the differentiation in vivo ability for detecting hiPS, undifferentiated iPSCs clone, about 1-2 × 10 are collected6Cell is injected into 6-8
In the hindlimb muscle of week old Male SCID mice, the formational situation of teratoma is observed.As shown in fig. 6, tumour is taken out after 8-12 weeks,
After conventional paraffin embedding slice, visible inside and outside triploblastica institutional framework is formed after HE dyeing.
(2), the liver cell directed differentiation culture of iPS cell origin
1, the recovery of iPS cell: from liquid nitrogen container from taking out cell, the constant-temperature thawing in 37 DEG C of constant water bath box immediately, from
The heart abandon supernatant remove dimethyl sulfoxide, be added ES culture medium be resuspended cell precipitation, planted ready mice embryonic at
It on fiber feeder cells (feeder), changes the liquid once daily, passage is primary weekly.
2, the culture of iPS cell: after detection is determined without mycoplasma, germ contamination, the iPS cell gram to grow fine is chosen
Grand, hand cut is passaged on new feeder cells, the cell after passage, is changed the liquid once per hour for 24 hours, is observed cell daily
Upgrowth situation.
3, the induction differentiation of iPS cell: when iPS cell when best in growth conditions on Feeder, by cell craft
Cutting, which is passaged in the culture dish for being coated with Matrigel glue in advance, cultivates.In mTESR cultivating system culture cell 2-4 days, change
Induced medium was replaced later with the culture of mouse fibroblast cell conditioned medium one day.Specific Induction Process is as follows:
First stage (3-6 days) is that iPS cell is induced to limited entoderm: the 1-2 days induced mediums be to
Everybody activin A and Wnt3a are added in basal medium RPMI-1640, wherein the use concentration of everybody activin A and Wnt3a
Respectively 5-300ng/ml and 20-100ng/ml, preferred concentration are respectively 100ng/ml and 25ng/ml；The 3-4 days culture mediums
To add everybody activin A and fetal calf serum into basal medium RPMI-1640, the wherein final concentration of 10- of human activin A
200ng/ml, the final concentration of 0.2-2% of fetal calf serum；The 5-6 days culture mediums are to add into basal medium RPMI-1640
Everybody activin A and fetal calf serum, the wherein final concentration 10-200ng/ml of everybody activin A, fetal calf serum it is final concentration of
0.2-2%；The dead cell of induction a few days ago is more, in order to avoid dead cell influences the progress of Induction Process, is carrying out replacement training
It exhausts as far as possible when supporting base and culture medium and cleans one to twice with RPIM-1640, then replace culture medium.
Second stage (6-12 days) is to liver system directed differentiations: culture medium used in 1-4 days is to basis before second stage
The fetal calf serum of 15-100ng/ml keratinocyte growth factor and final concentration of 2-3% is added in culture medium KO/DMEM.?
In the incubation of two-stage, cell quantity should take the circumstances into consideration to add according to the quantity of cell at this time compared with a few days ago there is biggish change
Add culture medium, to guarantee the normal growth of cell；Hereafter 5-12 days culture mediums are to add serum to basal medium KO/DMEM
Substitute (SR), L-Glutamine, nonessential amino acid, beta -mercaptoethanol and dimethyl sulfoxide, the primary training of replacement in every 24 hours
Base is supported, wherein final concentration of the 20% of serum substitute, the final concentration of 1-3mM of L-Glutamine, the end of nonessential amino acid
Concentration is 0.1%-15%, the final concentration of 0.1-0.12mM of beta -mercaptoethanol, the final concentration of 0.2-2% of dimethyl sulfoxide.
Three phases (6-10 days) are to promote the hepatocyte maturation stage: culture medium is to add into basal medium L-15
Cow's serum, hepatocyte growth factor, tumour inhibitor, dexamethasone, L-Glutamine, every 24 hours one subcultures of replacement, wherein
Final concentration of the 10% of fetal calf serum, the final concentration of 10-300ng/ml of hepatocyte growth factor, the final concentration of 5- of tumour inhibitor
150ng/ml, final concentration of 0.05-1.2 μM of dexamethasone, the final concentration of 2-3mM of L-Glutamine.
Entire Induction Process lasts 15-28 days.
(3) identification of liver cell
1, the observation of cell Induction Process differentiation situation: from the daystart of induction, observation is thin under the microscope daily
The growth conditions of born of the same parents, adherent situation, differentiation situation, iPS cell nucleocytoplasmic ratio is high, and cell arrangement is close, in irregular spherical colony
Growth.Induction starts rear cell and morphological change takes place, and the cell of induction 3 days enters the entoderm stage, and cell becomes flat
Flat is in oval；Cell initially enters the liver cell specialization stage after into second stage induction, and it is obvious that cell gradually becomes boundary
Polygonal；Phase III cell initially enters the liver cell stage of ripeness, and cell colony is finer and close, and cell volume increases,
In cube, endochylema is abundant, has one or more nucleus, and kernel is obvious.As shown in fig. 7, cultivating obtained cell and liver
Parenchyma more approaches.
2. the expression of Immunofluorescence test AFP, ALB: cell is used at room temperature 4wt% (i.e. mass concentration, it is the same below
Reason) paraformaldehyde fixes 15 minutes, and then the Triton X100 of 0.2v/v% permeates 15 minutes, with 4v/v% normal goats blood
Clear closing 1 hour, primary antibody is diluted in confining liquid, and primary antibody is incubated at 4 DEG C and is stayed overnight.Incubation at room temperature secondary antibody one hour.DAPI is added
It redyes 5 minutes.In fluorescence microscopy microscopic observation positive cell, as a result as shown in Fig. 8.
3.RT-QPCR: the cell for collecting each induction period last day is detected, and TRIzol dissolves extract RNA.Root
Reverse transcription is carried out according to operating instruction application Reverse Transcriptase kit, each sample contains the RNA of 1 μ g.PCR reaction system includes 0.2 μ
The PCR Green Mix of the cDNA of l, 10 μ l, 7.8 μ l water, each 1 μ l of 1 μM of forward and reverse primer verify the sequence difference of primer pair
See SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ
ID NO.11-12、SEQ ID NO.13-14、SEQ ID NO.15-16、SEQ ID NO.17-18、SEQ ID NO.19-20、
SEQ ID NO.21-22, SEQ ID NO.23-24 and SEQ ID NO.25-26.Circulation carry out it is as follows: 95 DEG C 5 minutes, then
45 circulation 95 DEG C 10 seconds, 58 DEG C 10 seconds, 72 DEG C 10 seconds, as a result as shown in Fig. 9.
The hepatic precursor cells of 4.iPS cell origin absorb and release ICG detection: ICG are dissolved in dimethyl sulfoxide,
Concentration is 5mg/ml, is then diluted to final concentration of 1mg/ml with differential medium.At the 20th day of cell induction, cell is existed
Routine culture 90 minutes in culture medium containing ICG after being rinsed with phosphate buffer, observe and record cell by microscope
Then the absorbing state of ICG changes ordinary culture medium culture 6 into and more than hour detects the release conditions of ICG, as a result such as attached drawing
Shown in 10.
5. the synthesis of periodic acid-Xue Fushi reaction detection glycogen: 20 days hepatic precursor cells 4wt% polies will be induced
The Triton X100 of formaldehyde and 1v/v% processing, then according to periodic acid-Xue Fu Albert'stain Albert system, (Sigma-Aldrich is public
Department, Pittsburgh, USA) specification dyed, as a result as shown in Fig. 11.
6. low-density lipoprotein intake experiment: cell is being contained 10 μ g/ml Alexa-Flour 488-labeled LDL
Serum free medium in cultivate 3 to 4 hours.Then it is redyed with DAPI, fluorescence microscopy microscopic observation fluorescence, as a result such as attached drawing
Shown in 12.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (2)
1. a kind of method that inductive pluripotent stem cells Induction of committed differentiation is liver cell, which comprises the steps of:
Step S1 provides or prepares inductive pluripotent stem cells；
Step S2, first stage culture, totally 3~6 days, in the first stage the 1-2 days, will be at the beginning of the inductive pluripotent stem cells
Culture begin in the RPMI-1640 culture medium added with human activin A and Wnt3a, and the culture under fetching in the first stage
RPMI-1640 culture medium of the replacement added with human activin A and fetal calf serum, makes the inductive pluripotent stem cells to limit in journey
Qualitative entoderm induction differentiation；Human activin A described in the RPMI-1640 culture medium added with human activin A and Wnt3a
Concentration be 100ng/ml, the concentration of the Wnt3a is 25ng/ml；It is described added with human activin A and fetal calf serum
The concentration of human activin A described in RPMI-1640 culture medium is 10-200ng/ml, and the volumetric concentration of the fetal calf serum is
0.2-2%；First stage induction dead cell a few days ago is more, in order to avoid dead cell influences the progress of Induction Process, into
It goes and exhausts culture medium when replacing culture medium as far as possible and clean one to twice with RPIM-1640, then replace culture medium；
Step S3, second stage culture, total 6-12 days, at the beginning of first 1-4 days of second stage, the cell that step S2 is cultivated
Culture begin in the KO/DMEM culture medium added with keratinocyte growth factor and fetal calf serum, and hereafter in second stage
It is changed in incubation sub- added with serum substitute, L-Glutamine, nonessential amino acid, beta -mercaptoethanol and dimethyl
The KO/DMEM culture medium of sulfone induces the cell of culture to liver system cell directional and breaks up；It is described to be added with Keratiocyte growth factor 1
The concentration of keratinocyte growth factor described in the KO/DMEM culture medium of son and fetal calf serum is 15-100ng/ml, the tire ox
The volumetric concentration of serum is 2-3%；It is described to be added with serum substitute, L-Glutamine, nonessential amino acid, beta -mercaptoethanol
And the volumetric concentration of serum substitute described in the KO/DMEM culture medium of dimethyl sulfoxide is 20%, the L-Glutamine
Concentration is 1-3mM, and the mass concentration of the nonessential amino acid is 0.1-15%, and the concentration of the beta -mercaptoethanol is 0.1-
0.12mM, the volumetric concentration of the dimethyl sulfoxide are 0.2-2%；In the incubation of second stage, cell quantity compared with
A few days ago there is biggish change, should be taken the circumstances into consideration to add culture medium at this time according to the quantity of cell, to guarantee the normal growth of cell；
Step S4, in the phase III, the cell culture that step S3 is cultivated is being added with fetal calf serum, hepatocyte growth factor
Son, tumour inhibitor, dexamethasone and L-Glutamine L-15 culture medium in cultivate 6-10 days, promote hepatocyte maturation, every 24 is small
Mono- subculture of Shi Genghuan；It is described to be added with fetal calf serum, hepatocyte growth factor, tumour inhibitor, dexamethasone and L- glutamy
The volumetric concentration of fetal calf serum described in the L-15 culture medium of amine is 10%, and the concentration of the hepatocyte growth factor is 10-
300ng/ml, the concentration of the tumour inhibitor are 5-150ng/ml, and the concentration of the dexamethasone is 0.05-1.2 μM, the L- paddy
The concentration of glutamine is 2-3mM；
Entire Induction Process lasts 15-28 days.
2. the method that inductive pluripotent stem cells Induction of committed differentiation as described in claim 1 is liver cell, which is characterized in that
In the step S1, it is imported using virus infection, plasmid transfection, mRNA, miRNA is imported or chemical molecular addition manner will lure
Inducement imports in human cell, and the human cell is induced to be divided into inductive pluripotent stem cells；The inducible factor includes
OCT4, SOX2, NANOG and Lin28.
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