CN103374546B - Hepatic parenchymal cells and its preparation, identification and application process - Google Patents
Hepatic parenchymal cells and its preparation, identification and application process Download PDFInfo
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Abstract
The invention discloses a kind of hepatic parenchymal cells and its preparation, identification and application processes.The method that offer of the present invention prepares hepatic parenchymal cells, includes the following steps：1) pluripotent stem cell culture is obtained into endoderm cell；2) endoderm cell that will be obtained from step 1) cultivates the original intestinal tube cell of acquisition；3) the original intestinal tube cell culture that will be obtained from step 2) obtains hepatoblast；It is characterized in that：The cell that will be obtained from step 2) is passed on, and is transferred to and is cultivated in the liver cell inducing culture I containing fibroblast growth factor (FGF) and bone morphogenetic protein (BMP), obtains hepatoblast；4) the hepatoblast culture that will be obtained from step 3) obtains ripe hepatic parenchymal cells.The invention further relates to the hepatic parenchymal cellses prepared according to the method.Hepatic parenchymal cells prepared by the present invention has higher metabolic activity more in the past, and preparation efficiency is high, has a extensive future.
Description
Technical field
The present invention relates to hepatic parenchymal cells and its preparation, identification and application processes.
Background technology
Liver plays an important role during human body metabolism, including Glycogen synthesis, urea synthesizing, fat metabolism tune
Control and drug detoxication etc..The damage of liver cell includes hepatitis, liver cancer, and hepatic sclerosis can cause pole safely to human life
Big harm.It solves the problems, such as this it is necessary to which a large amount of liver cells can be obtained in vitro, is transplanted using these cells.
In various cell deriveds, people's pluripotent stem cell (pluripotent stem cells, PSCs), including human embryo stem cell
(embryonic stem cells, ESC) and people's inducing pluripotent stem cells (induced pluripotent stem
Cells, iPSC), most application potential, human embryo stem cell have the ability of infinite multiplication and the totipotency of differentiation, are closing
Various cell types (Thomson the et al., Science.1998Nov 6 of human body can be divided under conditions of suitable；282
(5391):1145-7), it is the preferable donor cell sources for the treatment of various degenerative diseases.Furthermore, it is contemplated that hepatotoxicity is
One of sixty-four dollar question in new drug design and detection, substantial amounts of hepatic parenchymal cells source can greatly promote and establish new drug
Screening strategy.
By research during the last ten years, at present people induction people's pluripotent stem cell in terms of hepatic parenchymal cells differentiation
Through obtaining certain progress.We are inducing human stem cell to obtain larger breakthrough in terms of liver cell differentiation within 2007
(Cai et al.,Hepatology.2007May；45(5):1229-39).We are first by entire atomization according to body
The pattern of interior development is divided into several stages.Embryonic stem cell is effectively induced into entoblast first with Activin A.
Referring next to the signal path developed in vivo, two signal paths of FGF and BMP are activated, endoderm cell is promoted to be changed into early stage
Liver mother cell.Then the further differentiation of liver mother cell is promoted by the stimulation of HGF, finally utilizes OSM and Dexamethasone
Promote the maturation of hepatic parenchymal cells.The scheme so progressively induced can efficiently generate hepatic parenchymal cells very much, obtained liver
Dirty cell accounts for 70% or so of all inducers.Also, the cell in these human embryo stem cell sources expresses the special mark of liver
Will gene and participate in liver metabolism function enzyme, can Albumin Secretion, store glycogen, absorb and be metabolized indocyanine green, point
Low-density lipoprotein is secreted, and can be infected by HCV pseudovirus.The liver cell of transplant experiment finder's derived from embryonic stem cells can
To be maintained in the Mouse Liver of immunodeficiency type at least two weeks.These experiments prove that human embryo stem cell efficient can break up
Into the liver parenchymal cell with certain function, differentiation of the embryonic stem cell to liver cell is pushed to a new height.
Then, we also further prove that the differentiation scheme is readily applicable to people's inducing pluripotent stem cells and breaks up to hepatic parenchymal cells
(Song et al.,Cell Research 2009Nov；19(11):1233-42).Recently, other experimental groups also report
Efficient differentiation of human pluripotent stem cell obtains the experimental program of liver cell.Stephen A.Duncan, Ludovic Vallier
The liver cell of Albumin Secretion is obtained for using similar substep abductive approach with the groups such as Ian Wilmu, and can be just
Step detects mature hepatocytes expression cell cytochrome p 450 enzyme.In addition, Hesham Basma et al. are special using surface of hepatocytes
Different mark ASGPR sortings, which purify the liver cell come from people ESC differentiation and are transplanted to Mice Body interior energy, sees that apparent people source is thin
Born of the same parents rebuild mouse liver and secrete the albumin in people source.
Although differentiation of people's pluripotent stem cell to hepatic parenchymal cells has been achieved for some progress, at present through dividing
It is still incomplete, it is necessary to further optimize, the liver that people's pluripotent stem cell is promoted to break up to change the hepatic parenchymal cells obtained
Cell is finally ripe so that the hepatic parenchymal cells in people's pluripotent stem cell source really has availability.Although as previously mentioned,
Break up obtained hepatic parenchymal cells and expressed substantial amounts of functional gene, but if with primary separated people's adult liver parenchyma
Cell is compared, and also has certain difference：It is that the secretory volume of albumin cannot reach the level of adult hepatic always first, even
There is the difference on the order of magnitude.Secondly, the function of adult hepatic is extremely complex, for function of detoxification, liver expression hundreds of not
With the detoxication enzyme of hypotype, there is the metabolism of the first phase and the second phase.There is presently no prove to obtain under any differentiation condition
Hepatic parenchymal cells have these complete functions of detoxification.Again, under suitable model, people's adult hepatic cell can be high
Being integrated into Mouse Liver for efficiency forms humanization liver, and integration efficiency alreadys exceed 80% at present.But in noble cells
In liver transplant experiment, the integration of indivedual human archeocytes can only be detected in individual mice at present, efficiency is very low.
Therefore, there is an urgent need to a kind of new differentiation scheme, the differentiation of people's pluripotent stem cell is become more ripe, is had more
The hepatic parenchymal cells of function, to meet in studies and clinical application the needs of to the cell.Simultaneously, it is also desirable to which searching out one kind has
The identification method of effect, can distinguish these more ripe hepatic parenchymal cellses and those do not have detoxicating activity, albumin secretory volume
Relatively low liver cell, in order to which the cell is detected and is identified in studies and clinical application.
The content of the invention
The object of the present invention is to provide a kind of hepatic parenchymal cells and its preparation, identification and application processes.
The present invention relates to following many aspects：
1st, the method for preparing hepatic parenchymal cells, includes the following steps：
1) pluripotent stem cell culture is obtained into endoderm cell；
2) endoderm cell that will be obtained from step 1) cultivates the original intestinal tube cell of acquisition；
3) the original intestinal tube cell culture that will be obtained from step 2) obtains hepatoblast；
It is characterized in that：The cell that will be obtained from step 2) is passed on, and is transferred to and is being contained Desmocyte growth factor
It is cultivated in the liver cell inducing culture I of sub (FGF) and bone morphogenetic protein (BMP), obtains hepatoblast；
4) the hepatoblast culture that will be obtained from step 3) obtains ripe hepatic parenchymal cells,
Wherein described pluripotent stem cell is that the pluripotency of the embryonic stem cell or induced synthesis obtained from commercial channels is done
Cell.
1 in above-mentioned steps) it, 4) may be referred to the specific open of the present invention and combine the prior art (such as background technology
Described in those) introduction easily implement.
2nd, the method according to 1, wherein the step 2) includes：The cell that will be obtained from step 1) is transferred to containing into fibre
Tie up the original intestinal tube induction of the micromolecular inhibitor of Porcine HGF (FGF) and transforming growth factor (TGF-b) signal path
It is cultivated in culture medium, so as to obtain original intestinal tube cell.
3rd, the method according to 1 or 2, wherein the original intestinal tube inducing culture includes Desmocyte growth factor
The micromolecular inhibitor SB431542 of son -7 and transforming growth factor signal path；Wherein described liver cell inducing culture I bags
Containing fibroblast growth factor-7, bone morphogenic protein -2 and bone morphogenic protein -4.
4th, the method according to any one of 1-3, wherein step 3) further comprise that detection obtained into liver
The expression of the PROX1 and HNF6 of cell, the efficiency for preferably generating the hepatoblast of coexpression HNF4A and PROX1 are more than 60%.
5th, the method according to any one of 1-4, wherein the pluripotent stem cell is the embryo obtained from commercial channels
Tire stem cell or the pluripotent stem cell of induced synthesis, preferably mammalian cell, more preferably mouse or people's cell, it is optimal
It chooses cell, wherein, the embryonic stem cell obtained from commercial channels is preferably following any cell lines：BG01,BG02,
BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ES06, TE03, TE32, TE33, TE04,
TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14；The number that the number is NIH.
6th, the hepatic parenchymal cells that the method according to any one of 1-5 obtains.
7th, the hepatic parenchymal cells according to 6, the cell Albumin Secretion, secreted albumin level reach people's original
For the level of adult hepatic parenchymal cells Albumin Secretion, it is preferable that the cell also have selected from urea synthesis, storage glycogen,
Cytochrome P 450 enzymes are generated, carry out a phase, phase II metabolic, one or more functions in three-phase transhipment.
8th, according to the hepatic parenchymal cells described in 6 or 7 preparing artificial liver, drug metabolism study, drug toxicity survey
Application in examination or drug screening.
9th, the culture medium of original intestinal tube cell is prepared by pluripotent stem cell, the culture medium is to be given birth to containing fibroblast
The original intestinal tube inducing culture of the micromolecular inhibitor of the long factor (FGF) and transforming growth factor (TGF-b) signal path, it is excellent
Original intestinal tube inducing culture described in selection of land includes the small of fibroblast growth factor-7 and transforming growth factor signal path
Molecule inhibitor SB431542.
10th, the culture medium of hepatic parenchymal cells is prepared by pluripotent stem cell, the culture medium is to be given birth to containing fibroblast
The long factor (FGF) and the liver cell inducing culture I of bone morphogenetic protein (BMP), preferably described liver cell Fiber differentiation
Base I includes fibroblast growth factor-7, bone morphogenic protein -2 and bone morphogenic protein -4.
11st, the method for hepatic parenchymal cells of the identification with metabolic function, it is characterised in that the described method includes detection PROX1
Or the step of expression of the HNF6 in hepatic parenchymal cells.In an embodiment of this aspect of the present invention, CYP can be used
Hepatic parenchymal cells is marked in the fluorogenic substrate DBOMF of enzyme.
12nd, applications of the PROX1 or HNF6 in the kit for identifying the hepatic parenchymal cells with metabolic function is prepared.
In an embodiment of this aspect of the present invention, the fluorogenic substrate DBOMF of CYP enzymes can be included in the kit,
For hepatic parenchymal cells to be marked.
13rd, the method for hepatic parenchymal cells of the identification with metabolic function, it is characterised in that use the fluorogenic substrate of CYP enzymes
Hepatic parenchymal cells is marked in DBOMF.
14th, the fluorogenic substrate DBOMF of CYP enzymes is being prepared for the kit of hepatic parenchymal cells of the identification with metabolic function
In application.
Various aspects of the invention and its advantage are further as detailed below.
Hepatic parenchymal cells provided by the present invention, preferably by people's pluripotent stem cell (people PSC), including from business way
What the human embryo stem cell (people ESC) or pluripotent stem cell (people iPSC) differentiation of people's induction that footpath obtains obtained, secretion is higher
The albumin of content, and there is urea synthesis, storage glycogen, generate cytochrome P 450 enzymes, one phase of progress, phase II metabolic, three
The mutually functions such as transhipment.
The method that the present invention prepares hepatic parenchymal cells can include following exemplary step：
1) people's pluripotent stem cell is cultivated on entoderm inducing culture I；
2) cell that step 1) obtains is cultivated on entoderm inducing culture II；
3) cell that step 2) obtains is cultivated on entoderm inducing culture III, obtains endoderm cell；
4) endoderm cell that step 3) obtains cultivates on original intestinal tube inducing culture, obtains original intestinal tube cell；
5) the original intestinal tube cell obtained to step 4) passes on, and is cultivated on liver cell inducing culture I；
6) cell that step 5) obtains is cultivated on liver cell inducing culture II；
7) cell that step 6) obtains is cultivated on liver cell inducing culture III, obtains hepatic parenchymal cells.
The entoderm inducing culture I is the bovine serum albumin(BSA) component V containing mass percentage 0.01%-1%
With the basal cell culture medium of 5-500ng/ml human activins-A；Wherein, the content of bovine serum albumin(BSA) component V is preferably
0.05%；The content of human activin-A is preferably 100ng/ml；
The entoderm inducing culture II is the bovine serum albumin(BSA) component containing mass percentage 0.01%-1%
Insulin-transferrins-sodium selenite mix supplement liquid of V, volumn concentration 0.1%-10% and 5-500ng/ml people
The basal cell culture medium of activin-A；Wherein, the content of bovine serum albumin(BSA) component V is preferably 0.05%；Human activin-A
Content be preferably 100ng/ml；The content of insulin-transferrins-sodium selenite mix supplement liquid is preferably 0.1%；
The entoderm inducing culture III is the bovine serum albumin(BSA) component containing mass percentage 0.01%-1%
Insulin-transferrins-sodium selenite mix supplement liquid of V, volumn concentration 0.1%-10% and 5-500ng/ml people
The basal cell culture medium of activin-A；Wherein, the content of bovine serum albumin(BSA) component V is preferably 0.05%；Human activin-A
Content be preferably 100ng/ml；The content of insulin-transferrins-sodium selenite mix supplement liquid is preferably 1%；
The original intestinal tube inducing culture is to contain 1-100ng/ml human fibroblastic growth factors -7,1-100mM
The bovine serum albumin(BSA) component V's and volumn concentration 0.1%-10% of SB431542, mass percentage 0.01%-1%
The basal cell culture medium of insulin-transferrins-sodium selenite mix supplement liquid；Wherein, human fibroblastic growth factor
The content of son -7 is preferably 20ng/ml；The content of SB431542 is preferably 10mM；The content of bovine serum albumin(BSA) component V is preferred
For 0.05%；The content of insulin-transferrins-sodium selenite mix supplement liquid is preferably 1%；
The liver cell inducing culture I is to contain 1-100ng/ml human fibroblastic growth factors -7,1-100ng/
The base of ml people bone morphogenic protein -2,2-200ng/ml people bone morphogenic protein -4 and volumn concentration 0.1-10%B27 replenishers
Plinth cell culture medium；Wherein, the content of human fibroblastic growth factor -7 is preferably 20ng/ml；People's bone morphogenic protein -2
Content is preferably 20ng/ml；The content of people's bone morphogenic protein -4 is preferably 50ng/ml；The content of B27 replenishers is preferably 1%.
The liver cell inducing culture II is to contain 1-100ng/ml human hepatocyte growth factors, 2-200ng/ml people's bone
It is molded the basal cell culture medium of protein-4 and volumn concentration 0.1-10%B27 replenishers；Wherein human hepatocyte growth because
The content of son is preferably 20ng/ml；The content of people's bone morphogenic protein -4 is preferably 50ng/ml；The content of B27 replenishers is preferably
1%.
The liver cell inducing culture III is to contain 1-100ng/ml people tumour inhibitor-M and 0.01-10 μM of dexamethasone
Hepatocyte culture medium；The content of wherein people's tumour inhibitor-M is preferably 10ng/ml；The content of dexamethasone is preferably 0.1 μM.
Above-mentioned entoderm inducing culture I, entoderm inducing culture II, entoderm inducing culture III, original intestinal tube
Inducing culture, liver cell inducing culture I, liver cell inducing culture II, the pH of liver cell inducing culture III can be
Cultivate the conventional pH, such as pH7.2-7.6. of mammalian cell
The basal cell culture medium can be MEM, DMEM, BME, DMEM/F12, RPMI1640, William E or
Fischers culture mediums.
In the method, the pluripotent stem cell of human embryo stem cell or induction is cultivated in entoderm inducing culture I
1-3 days, be preferably 1 day；The cell that step 1) obtains is cultivated 1-3 days in entoderm inducing culture II, is preferably 1 day；Step
The rapid cell 2) obtained is cultivated 1-3 days in entoderm inducing culture III, is preferably 1 day；The entoderm that step 3) obtains is thin
Born of the same parents cultivate 1-5 days in original intestinal tube culture medium, are preferably 2 days；The original intestinal tube cell that step 4) obtains is after passage, in liver
It is cultivated 3-8 days in cell inducing culture I, is preferably 5 days；The cell that step 5) obtains is trained on liver cell inducing culture II
It supports 3-8 days, is preferably 5 days；The cell that step 6) obtains is cultivated 3-8 days on liver cell inducing culture III, is preferably 5 days,
Obtain hepatic parenchymal cells.
In the method, the step of being passed on original intestinal tube cell, the propagating method of original intestinal tube cell are further included
For by the original intestinal tube cell Accutase digestive juices (Millipore companies of the U.S.) or the pancreas enzyme -EDTA digestive juice (U.S.
Invitrogen companies) digestion, then cultivated on hepatocyte culture medium I.
By above-mentioned entoderm inducing culture I, above-mentioned entoderm inducing culture II, above-mentioned entoderm inducing culture
III, above-mentioned original intestinal tube culture medium, above-mentioned liver cell inducing culture I, above-mentioned liver cell inducing culture II and above-mentioned liver are thin
The training for being used to be prepared hepatic parenchymal cells by the pluripotent stem cell of human embryo stem cell or induction of born of the same parents' inducing culture III compositions
Foster base falls within protection scope of the present invention.
There is good cytochrome P 450 enzyme activity using the people's hepatic parenchymal cells for inventing acquisition herein, liver can be carried out
One phase, phase II metabolic and three-phase transhipment, functionally better than the liver cell broken up by other schemes in the past.By to obtaining
Hepatic parenchymal cells carry out identification and analysis, find we obtain cell not only Cytochrome P450 activity and albumin
Secretion etc. is functionally better than previous liver cell, it is often more important that in gene expression, expresses and was obtained in the past by other schemes
The key transcription factor PROX1 and HNF6 that the liver cell obtained is not expressed, so as to cause significantly improving functionally.
Description of the drawings
Fig. 1 is the preparation process schematic diagram of hepatic parenchymal cells.
Fig. 2 is the expression trend for preparing key gene during hepatic parenchymal cells.
After Fig. 3 is the first stage, entoderm marker gene in the cell expression embryo that immunofluorescence display differentiation obtains
SOX17, FOXA2 and GATA4, but primitive endoderm marker gene AFP is not expressed, illustrate that people's pluripotent stem cell has broken up
For endoderm cell in embryo.
After Fig. 4 is second stage, the cell of immunofluorescence display differentiation start to express original intestinal tube mark transcription because
Sub- HNF1B and HNF4A, while maintain the expression of entoderm marker gene SOX17.
After Fig. 5 is the phase III, the cell mass that immunofluorescence shows a group AFP positives occurs, and these are thin
Born of the same parents express HNF4A, HNF6, PROX1 and TBX3 simultaneously.
Fig. 6 is quantitative PCR result.It shows compared with the cell (d5) of differentiation in the 5th day, whether was carried out at the 6th day at passage
Manage (R) and after whether differentiation in the 6-10 days adds in cell factor (F) in the process, the situation of key gene expression.
Fig. 7 breaks up for Flow cytometry analysis to after the 10th day, PROX1 and the double positive cell proportions of HNF4A.Not
The stem cell (day 0) of differentiation is used as negative control.
After Fig. 8 is fourth stage, immunofluorescence shows cell mass coexpression AFP, PROX1, HNF6 and HNF4A.
The stage, cell mass also begin to expression CEBPA and HNF1A, illustrate the cell of differentiation further to hepatocytic fate specialization.
Fig. 9 is to contain small oil droplet in the hepatic parenchymal cells being prepared.Red is specifically coloured for small oil droplet by oil red O.It is green
The expression for the CYP3A4 that color shows for immunofluorescence.It can be found that the region of CYP3A4 expression and the region one of oil red O colorings
It causes.
Figure 10 is the expression of various key transcription factors in the hepatic parenchymal cells being prepared.Green is CYP3A4's
Dyeing, identifies hepatic parenchymal cells, and red is the dyeing of transcription factor.
Figure 11 is the immunofluorescence dyeing of CYP3A4, CYP1A2, ALB and AAT.Most region is total in the cell of differentiation
Express ALB and CYP3A4 and CYP1A2, it is shown that differentiated efficiency.Meanwhile these cells also express ripe functional gene
AAT。
Figure 12 shows the efficiency that hepatic parenchymal cells is prepared for flow cytometry.Break up obtained hepatic parenchymal cells table
Up to special surface marker ASGPR.Simultaneously, additionally it is possible to which the substrate DBOMF of CYP3A4 is metabolized into green fluorescence product.
Figure 13 is to confirm that the substrate DBOMF of CYP3A4 is metabolized by the hepatic parenchymal cells being prepared under fluorescence microscope
Green fluorescence product.This metabolic process can be blocked by the specific inhibitor Ketoconazole (KC) of CYP3A4.
Figure 14 is the secretion that enzyme-linked immunization detects albumin (ALB) and α-antitrypsin.HPS, people's pluripotency are done carefully
Born of the same parents；F+R-, by cytokine induction but without the noble cells of passage processing acquisition；F+R+, by cytokine induction
And the noble cells obtained by passage processing；25w FL, 25 weeks fetal liver cells；AH, the primary hepatic parenchymal cells of adult.
Figure 15 is the Function detection of the hepatic parenchymal cells prepared.Left figure is PAS Coloration experiments；Right figure absorbs for indocyanine green
Experiment.
Figure 16 is to detect human albumin content in mouse after the transfer.
Figure 17 is that the hepatic parenchymal cells that GC-MS method obtains carries out a phase and the ability of phase II metabolic.
Figure 18 be in the hepatic parenchymal cells of preparation typical medicaments to cytochrome P 450 enzymes suppression curve.Upper figure,
Furafylline inhibits the curve of CYP1A2；Figure below, Ketoconazole inhibit the curve of CYP3A4.
Figure 19 is the three-phase transport activity detection of the hepatic parenchymal cells prepared.Green fluorescence shows, small molecule CLF (above)
Or after CDF (figure below) is added in culture medium, cut by esterase, fluorescence-causing substance is formed, is specifically secreted into liver cell
In surrounding cholangiole structure.
Figure 20 is a phase metabolic enzyme CYP3A4 and to turn in the liver cell obtained using the mode reported before the present invention
Record the expression of the factor PROX1 and HNF6.The hepatoblast stage in atomization, D figures and E is shown in wherein A-C figures
Figure was shown in the last hepatic parenchymal cells stage eventually.Notice that the cell of a small amount of CYP3A4 positives is confined to PROX1 or HNF6
In the cell mass of expression.
Figure 21 is when interfering the expression of HNF6 (A figures downlink) or PROX1 (B figures downlink) with shRNA, and immunofluorescence is shown
The expression of metabolic enzyme CYP3A4 is also lowered therewith.Interfere experiment (A the schemes and uplink of B figures) conduct of green fluorescent protein (EGFP)
Control.
Figure 22 be with shRNA interfere HNF6 or PROX1 expression when, quantitative PCR analysis show metabolic enzyme CYP3A4 and
The expression of ALB is also lowered therewith, but the expression of HNF4A is unaffected.Interfere the experiment conduct pair of green fluorescent protein (EGFP)
According to.
Figure 23 is the hepatic parenchymal Immunofluorescence test that the pluripotent stem cell of employment induction is prepared, and shows metabolic enzyme
CYP3A4 is co-expressed with transcription factor PROX1 or HNF6.
Specific embodiment
Method therefor is conventional method unless otherwise specified in following embodiments, and agents useful for same commercially obtains
.Wherein, bovine serum albumin(BSA) component V (Calbiochem companies of the U.S., 126579), and human activin-A (Activin A, it is beautiful
Peprotech companies of state, 120-14E), insulin-transferrins-sodium selenite mix supplement liquid (U.S. Invitrogen
Company, 51300-044), B27 replenishers (Invitrogen companies of the U.S., 12587-010), the hepatocyte culture medium HCM (U.S.
Lonza companies, CC-3198), human fibroblastic growth factor -7 (FGF7, Peprotech companies of the U.S., 100-19),
SB431542 (tocris companies of the U.S., 1614), people's bone morphogenic protein -2 (BMP2, Peprotech companies of the U.S., 120-02),
People's bone morphogenic protein -4 (BMP4, R＆D systems companies of the U.S., 314-BP), human hepatocyte growth factor (HGF, the U.S.
Peprotech companies, 100-39), people's tumour inhibitor-M (OSM, R＆D systems companies of the U.S., 295-OM), dexamethasone
(Dexamethasone, Sigma-Aldrich, D8893).DBOMF is purchased from Invitrogen companies, article number：
P2979 or P2974)
Fig. 1-2 2 in embodiment is the corresponding cell obtained by human embryonic stem cell H1 (number of NIH is WA01),
Figure 23 is to be obtained by the pluripotent stem cell (3U1, bibliography Song et al., Cell Research, 2009) of people's induction
Corresponding cell, respectively by human embryonic stem cell H7 (number of NIH is WA07), the human embryonic stem cell H9 (numbers of NIH
For WA09) and other people induce pluripotent stem cell (people iPSC) obtain corresponding cell it is essentially identical with Fig. 9-19, do not have
Essential difference.
First, the acquisition of hepatic parenchymal cells
Differentiation step can be divided into five steps, with reference to Fig. 1.
The first step (the 1-3 days)：People's multipotent stem cells are divided into endoderm cell
1st day：
Start within 2-3 days to induce after human embryo stem cell H1, H7 or H9 passage, the good cell of growth selection state is divided
Change experiment；
Human embryonic stem cell medium is discarded (to add 20% serum substitute in basic cell culture medium DMEM/F12
(Knock-out Serum Replacement, KSR, Invitrogen companies of the U.S., 10828028), 1%GlutaMAX is (beautiful
Invitroge companies of state, 35050-061), 0.1mM beta -mercaptoethanols (Invitrogen companies of the U.S., 21985-023), 1%
Nonessential amino acid (Non-essential Amino Acids) (Invitrogen companies of the U.S., 11140-076), 4ng/mL
Basic fibroblast growth factor (bFGF, Peprotech companies of the U.S., 100-18B)), it is washed 2 times with PBS；
Change entoderm inducing culture I.Entoderm inducing culture I is to add in bovine serum albumin in 1640 culture mediums
Bai Zufen V (Calbiochem companies of the U.S., 126579) and human activin-A (Activin A, Peprotech companies of the U.S.,
Culture medium 120-14E) obtained.The pH7.2-7.6 of the culture medium.In entoderm inducing culture I, bovine serum albumin(BSA) component
Final concentration of 0.05% (mass percentage), the final concentration of 100ng/ml of human activin-A (Activin A) of V.
2nd day：
The culture medium of yesterday is discarded, changes entoderm inducing culture II.Entoderm inducing culture II is in 1640 trainings
It supports and bovine serum albumin(BSA) component V (Calbiochem companies of the U.S., 126579), human activin-A (Activin A) is added in base
The training obtained with insulin-transferrins-sodium selenite mix supplement liquid (Invitrogen companies of the U.S., 51300-044)
Support base.The pH7.2-7.6 of the culture medium.In entoderm inducing culture II, bovine serum albumin(BSA) component V's is final concentration of
0.05% (mass percentage), the final concentration of 100ng/ml of human activin-A (Activin A), insulin-turn iron egg
Final concentration of 0.1% (volumn concentration) of in vain-sodium selenite mix supplement liquid.
3rd day：
The culture medium of yesterday is discarded, changes entoderm inducing culture III.Entoderm inducing culture III is 1640
Bovine serum albumin(BSA) component V, human activin-A (Activin A) and insulin-transferrins-selenous acid are added in culture medium
The culture medium that sodium mix supplement liquid (Invitrogen companies of the U.S., 51300-044) obtains.The pH7.2-7.6 of the culture medium.It is interior
In germinal layer inducing culture III, final concentration of 0.05% (mass percentage) of bovine serum albumin(BSA) component V, human activin-
The final concentration of 100ng/ml of A (Activin A), insulin-transferrins-sodium selenite mix supplement liquid it is final concentration of
1% (volumn concentration).
Second step (the 4-5 days)：Endoderm cell is divided into original intestinal tube cell
4th day：
The culture medium of yesterday is abandoned, PBS is washed three times.Change original intestinal tube culture medium.Original intestinal tube culture medium is in 1640 trainings
Support added in base bovine serum albumin(BSA) component V, human fibroblastic growth factor -7 (FGF7, Peprotech companies of the U.S.,
100-19), SB431542 (tocris companies of the U.S., 1614) and insulin-transferrins-sodium selenite mix supplement liquid
The culture medium that (Invitrogen companies of the U.S., 51300-044) is obtained.The pH7.2-7.6 of the culture medium.Original intestinal tube culture medium
In, final concentration of 0.05% (mass percentage) of bovine serum albumin(BSA) component V, the end of human fibroblastic growth factor -7
Concentration be 20ng/ml, SB431542 final concentration of 10mM, the end of insulin-transferrins-sodium selenite mix supplement liquid
Concentration is 1% (volumn concentration).
5th day：
The culture medium of yesterday is abandoned, changes original intestinal tube culture medium.
3rd step (the 6-10 days)：Original intestinal tube cell differentiation is hepatoblast
6th day：
Prepare the coated culture dishes of Matrigel：Matrigel (U.S. company BD, 354230) is melted on ice, by 1:
33 dilute in the DMEM/F12 culture mediums of ice precooling, are added in culture dish, measure fully do not had culture dish to be advisable.37 degree incubate
It educates and discards culture medium after sixty minutes, it is spare.
The cell of differentiation：Culture medium yesterday is abandoned, PBS is washed one time；With Accutase enzymes (Millipore companies of the U.S.,
SCR005) digested, 37 degree are incubated after five minutes, are terminated with the culture medium containing serum.Cell, which is gently dispelled, to be become
It is unicellular.1200, which leave the heart, collects cell for 3 minutes, and supernatant discarding is resuspended with liver cell inducing culture I, and counted.
Contain 2 × 10 by every ml culture mediums5Cell number is induced in the culture dish being coated with Matrigel with liver cell
Culture medium I is cultivated.
Liver cell inducing culture I is that B27 replenishers (U.S. Invitrogen public affairs are added in DMEM/F12 culture mediums
Department, 12587-010), GlutaMAX, nonessential amino acid, b- mercaptoethanols, human fibroblastic growth factor -7, people's bone into
Type albumen -2, the culture medium that people's bone morphogenic protein -4 obtains.The pH7.2-7.6 of the culture medium.In liver cell inducing culture I,
Final concentration of 1% (volumn concentration) of B27 replenishers；The final concentration of 20ng/ml of human fibroblastic growth factor -7；
The final concentration of 20ng/ml of people's bone morphogenic protein -2；The final concentration of 50ng/ml of people's bone morphogenic protein -4；Beta -mercaptoethanol
Final concentration of 0.1mM；Final concentration of 1% (volumn concentration) of nonessential amino acid；Final concentration of the 1% of GlutaMAX
(volumn concentration).
The 7-10 days every two days repetition following steps：
The culture medium of yesterday is abandoned, PBS is washed one time；
Liver cell inducing culture I is changed, is cultivated.
4th step (the 11-15 days)：Hepatoblast is divided into jejune hepatic parenchymal cells
The 11-15 days every two days repetition following steps：
The culture medium of yesterday is abandoned, PBS is washed one time；
Liver cell inducing culture II is changed, is cultivated.Liver cell inducing culture II is in DMEM/F12 culture mediums
Middle addition B27 replenishers (Invitrogen companies of the U.S., 12587-010), GlutaMAX, nonessential amino acid, b- sulfydryl second
Alcohol, human hepatocyte growth factor, the culture medium that people's bone morphogenic protein -4 obtains.The pH7.2-7.6 of the culture medium.Liver cell induces
In medium ii, final concentration of 1% (volumn concentration) of B27 replenishers；Human hepatocyte growth factor it is final concentration of
20ng/ml；The final concentration of 50ng/ml of people's bone morphogenic protein -4；The final concentration of 0.1mM of beta -mercaptoethanol；Non-essential amino
Final concentration of 1% (volumn concentration) of acid；Final concentration of 1% (volumn concentration) of GlutaMAX.
5th step (the 16-20 days)：Jejune hepatic parenchymal cells is further divided into ripe hepatic parenchymal cells
Following steps are repeated daily within the 16-20 days：
The culture medium of yesterday is abandoned, PBS is washed one time；
Liver cell inducing culture III is changed, is cultivated.Liver cell inducing culture II is in hepatocyte culture medium
The culture medium that people tumour inhibitor-M and dexamethasone obtain is added in HCM (Lonza companies of the U.S., CC-3198).Liver cell induction training
It supports in base III, the final concentration of 10ng/ml of people's tumour inhibitor-M；Final concentration of 0.1 μM of dexamethasone.
2nd, in hepatic parenchymal cells atomization differentiation efficiency and cellularity identification
We have carried out the cell generated in atomization the identification (Fig. 2) of gene expression.Dryness gene OCT4 and
SOX2 high expression in embryonic stem cell, differentiation is then rapid after starting to be declined, and is formed to hepatoblast and the stage is then detected later
Less than the expression of gene.SOX17, FOXA2 and GATA4 high expression since the entoderm stage.HNF1B and HNF4A is in original intestines
There is significant up-regulation in the pipe stage.And hepatoblast stage, the high expression of AFP, PROX1, HNF6 and TBX3 have then indicated cell
Warp-wise liver lineage specialization.In the immature hepatic parenchymal cells stage, the up-regulation of CEBPA and HNF1A show that cell is further
It is ripe.In the last stage eventually, the high expression of cell ripe functional gene AAT, ALB and CYP3A4 illustrate that cell is finally divided into
Ripe hepatic parenchymal cells.
We make a concrete analysis of each step in atomization below：
Human embryo stem cell breaks up first as endoderm cell.After activin-A is added in, the expression of SOX17 is rapid
Up-regulation, while the expression of the marker gene OCT4 and SOX2 of human embryo stem cell are remarkably decreased (Fig. 2, Fig. 3).Then, entoderm
Cell is made by fibroblast growth factor-7 and the joint of the inhibitor SB431542 of Activin/TGF-b signal paths
With differentiation becomes the original intestinal tube cell of expression HNF1B and HNF4A.Make in fibroblast growth factor-7 and SB431542
After two days, HNF1B and HNF4A have significantly raised (Fig. 2, Fig. 4) in the expression of mRNA and protein level.
In the phase III, original intestinal tube cell carries out specialization to liver lineage.In pervious report, usually pass through adjusting
BMP and FGF signal paths carry out the specialization of liver destiny.In our current research, in order to obtain the complete hepatoblast of differentiation, we
It is innovative to be attempted using PROX1 and HNF6 for mark in differential medium, growth factor etc. etc., it is final it is definite most
Excellent condition is according to 2 × 10 by intestinal tube cell5/ milliliter density is layered on again on the coated culture dishes of Matrigel, and add in people into
Fibroblast growth factor -7, people's bone morphogenic protein -2 and people's bone morphogenic protein -4 are simultaneously cultivated 5 days.In such a situa-tion, go out
The hepatoblast of a group expression PROX1 and HNF6 is showed.These cells also express AFP and HNF4A (Fig. 5) simultaneously.By quantitative
PCR is analyzed, it has been found that only in inducement signal (addition growth factor) and in the case that succeeding generations exist simultaneously, can just be had
The expression (Fig. 6) of the induction PROX1 and HNF6 of effect.This explanation, the process simulation of the passage letter of Immune inducing in vivo PROX1 and HNF6
Number, and with BMP and FGF signal path parallel actions, together induction of the generation of hepatoblast.In order to further identify these
The hepatoblast of PROX1 and HNF6 is expressed, we also have detected some other played in hepatoblast destiny decision process again
The transcription factor to be acted on.Quantitative PCR and immunofluorescence the results show that most cell is also expressed in hepatoblast clone
TBX3, FOXA2 and GATA4 (Fig. 1 and Fig. 6).The above result illustrates that we are broken up to obtain by stem cell in vitro jointly
Hepatoblast and in vivo hepatoblast it is very similar.It is analyzed by intracellular streaming technology, in entire differentiated system,
The efficiency for generating the hepatoblast of coexpression HNF4A and PROX1 is about 63.7% (Fig. 7).
In fourth stage, hepatoblast continues in the culture medium containing hepatocyte growth factor and people's bone morphogenic protein -4
It is cultivated, so as to further to hepatic parenchymal cells destiny specialization.These hepatoblast clonal expansions, and maintain HNF4A,
The expression of PROX1 and HNF6.The stimulation of hepatocyte growth factor is have passed through, they also start to express hepatic parenchymal cells marker gene
CEBPA and HNF1A (Fig. 1 and Fig. 8).Except the appearance of CEBPA and HNF1A, the expression of HNF4A and ALB also lasting rising
(Fig. 1), illustrates that these cells have further carried out specialization to hepatic parenchymal cells destiny.
In terminal stage, differential medium is converted to liver cell inducing culture III by us, and not only its basic liver is real
Cell plastid culture medium HCM is more suitable for the growth of mature cell, but also contains mature factor Ziren tumour inhibitor-M and dexamethasone.
After induction 5 days, the cell clone of differentiation becomes flat, and small oil droplet (Fig. 9) is contained in some cells.We are further
Have detected the expression of liver transcription factor and functional gene.In the latter stage of differentiation, extensive region can detect in liver cell population
The expression of transcription factor PROX1, HNF6, HNF1A, CEBPA, HNF4A, FOXA2, HNF1B and CEBPB, and it is thin with liver parenchyma
Born of the same parents' marker gene CYP3A4 contaminates altogether；And (Figure 10) has then significantly been lowered in the expression of TBX3 and GATA4.These results in vivo just
The growth course of normal hepatic parenchymal cells matches.Further, also co-expressed while most noble cells expression ALB
CYP3A4, CYP1A2 and AAT (Figure 11).Flow Cytometry Analysis the result shows that, about 70% cell is being expressed
The substrate DBOMF of CYP3A4 can also be metabolized to fluorescence production while the surface marker molecule ASGPR of ripe hepatic parenchymal cells
Object (Figure 12).We also with immunofluorescence observation confirm metabolism DBOMF's as a result, also, this metabolic process be can be by
(Figure 13) that the specific inhibitor ketoconazole of CYP3A4 is blocked.
3rd, the function for the hepatic parenchymal cells that differentiation obtains
There is normal liver parenchyma cell in order to illustrate the hepatic parenchymal cells obtained in the present invention by differentiation, I
Into a series of Function detection.By enzyme-linked immunization it was found that the hepatic parenchymal cells that differentiation obtains secretes a large amount
Albumin A LB and α-antitrypsin AAT (Figure 14).The level of Albumin Secretion has reached 15.9 ± 2.8 every milligram of micrograms
Total protein is daily similar with the result of the primary adult hepatic parenchymal cells of people, and is significantly higher than the level of 25 weeks fetal liver cells of people.Such as
Fruit is not standardized on total protein content, and it is daily that the content of albumin in the medium is up to 5.1 ± 0.9 micrograms per millilitres, high
The attainable level of noble cells Albumin Secretion in report.The secretory volume level of α-antitrypsin can also be with adult liver
The secretion level of parenchyma is similar.In addition, we also prove the liver parenchyma of the expression CYP3A4 of differentiation by the dyeing of oil red O
Contain small oil droplet (Fig. 9) in cell；Being dyed by PAS proves that these cells can store glycogen；By being incubated altogether with indocyanine green
The experiment educated proves that these cells have the ability (Figure 15) for absorbing these dyestuffs.Noble cells is injected into newborn mice liver
In, after 4 weeks we can also detect the presence of human albumin in mice serum, it was demonstrated that these cells, which also have, is integrated into liver
Ability (Figure 16) in parenchyma.
We go back emphasis and have detected metabolic function possessed by these cells.Utilize the probe of hypospecificity and chromatography matter
Combination analysis is composed, we have detected in phase metabolism and are tied in each hypotype of FscP and phase II metabolic
The activity of synthase, and 25 weeks fetal liver cells of employment and adult hepatocytes are as control (Figure 17).In Human embryo development,
CYP3A4,2C9,2E1, UGT and SULT are usually in 4-9 middle of the month starting expression.In the hepatic parenchymal cells broken up at us, although
The expression of CYP3A4,2C9,2E1 and phase II metabolic enzyme is also less than primary adult hepatic, but is above or similar to fetal liver cell
Enzyme activity is horizontal.CYP2C19 and 1A2 is extremely low in embryonic period, embryonic phase expression during human hair is educated, and just starts to occur after birth.
In the hepatic parenchymal cells broken up at us, the two enzymes can have certain activity, and 25 weeks fetal liver cells as control are then
It is only able to detect the expression of relatively low background level.These results illustrate, break up the hepatic parenchymal cells of acquisition in the present invention and people encloses
The hepatic parenchymal cells in stage in term is similar.
Whether the hepatic parenchymal cells obtained to evaluate differentiation can be used in the research of drug interaction, we are further
Cytochromes are carried out using the classical inhibitor Furafylline of CYP3A4 classics inhibitor Ketoconazole and CYP1A2
The Inhibition test of P450 enzymes.It was found that with the raising for inhibiting agent dose, the hepatic parenchymal cells broken up is in the same terms
The generation of lower metabolite gradually decreases, and shows typical dose-dependent effects pattern (Figure 18).Broken up using us and obtained
The hepatic parenchymal cells obtained is measured, and the IC50 values of Ketoconazole are 0.29 μM, and the IC50 values of Furafylline are 19 μ
M, this is similar with the result obtained in the past in people's adult hepatic parenchymal cells.Therefore, by dividing in these description of test present invention
Change the hepatic parenchymal cells obtained and can be used for drug metabolism study.
In addition to a phase and phase II metabolic, we also have detected three-phase transport activity.We are by small molecule CDF
(carboxy-dichloro-fluorescein diacetate) or CLF (cholyl-lysl-fluorescein) are added in
In culture medium, both small molecules do not shine in itself, but are once absorbed by liver cell, will be cut by the esterase in liver cell,
Fluorescence-causing substance is formed, is specifically secreted into the transport function that liver in cholangiole structure, is indicated around liver cell.It can be with from Figure 19
See, the fluorescence-causing substance of CDF or CLF are evenly distributed in the periphery of noble cells, and only special in agglomerate sample liver cell
Different expression, the hepatic parenchymal cells that verification differentiation obtains also have three-phase transport function.
4th, PROX1 and HNF6 is vital to obtaining the hepatic parenchymal cells with metabolic function
It is basic although people can also obtain the cell of some hepatic parenchymal cells samples by breaking up before the present invention
Do not possess or only faint metabolic function.We are by having carried out these noble cells detailed detection.We send out
It is existing, in the hepatoblast stage for being expressed as representing with AFP, although these cells express the transcription factor of part hepatoblast,
Such as HNF4A, FOXA2, GATA4 and GATA6, but what the expression of PROX1 and HNF6 was missing from.And previous stem cell to
In the pertinent literature of hepatic parenchymal cells differentiation, table in the hepatoblast also generated without the two transcription factors in atomization
The report reached.In differentiation, end obtains the hepatic parenchymal cells stage eventually, the two transcription factors are in the overwhelming majority using the ALB positives as representative
Liver cell population in be also missing from.The cell of only only a few accidentally expresses PROX1 and HNF6, and only these expression
PROX1 and HNF6 cells just express metabolic function gene C YP3A4 (Figure 22).It is therefore believed that in stem cell liver cell point
It is divided into two cell masses during change, whether can be according to the expression of PROX1 and HNF6.Lack PROX1 and HNF6 expression
Cell mass is incomplete in destiny specialization, and metabolic function is also the absence of in differentiation eventually functioning cell caused by end.
In order to further prove that the two transcription factors are adjusting the importance of cytochrome P 450 enzymes appearance, we also exist
Hepatic parenchymal cells proposed by the present invention has carried out PROX1 and HNF6 gene expressions interference experiment (Figure 23) during preparing.I
Will be transduceed by slow virus for the shRNA of PROX1 or HNF6 into noble cells, and with quantitative PCR experiment and
Immunofluorescence experiment confirms PROX1 the HNF6 expression deletions in the cell for the corresponding shRNA that transduceed.And conduct pair
According to the expression of the cell PROX1 and HNF6 of the shRNA for being directed to green fluorescent protein that transduceed do not change then.Compared with the control,
The level for having lowered the mRNA of CYP3A4 in the cell of PROX1 or HNF6 expression is remarkably decreased, and antibody can not detect
The expression of the albumen of CYP3A4 illustrates that these cells can not break up the hepatic parenchymal cells as CYP3A4 expression again.And in addition
The expression of the marker gene HNF4A of one liver cell is then unaffected.These results further confirm PROX1 and HNF6
Play an important roll in hepatic parenchymal cells function maturation is adjusted.
Claims (14)
1. preparing the method for hepatic parenchymal cells, include the following steps：
1) pluripotent stem cell culture is obtained into endoderm cell；
2) endoderm cell that will be obtained from step 1) is transferred in original intestinal tube inducing culture and is cultivated, and obtains original intestinal tube
Cell；
3) cell that will be obtained from step 2) is passed on, and is transferred to and is being contained fibroblast growth factor-7, bone shaping egg
In vain -2 and bone morphogenic protein -4 liver cell inducing culture I in cultivate, the table of the PROX1 and HNF6 of the obtained cell of detection
It reaches, obtains the hepatoblast of expression PROX1 and HNF6；
4) the hepatoblast culture that will be obtained from step 3) obtains ripe hepatic parenchymal cells,
Wherein described pluripotent stem cell is inducing pluripotent stem cells, i.e., iPS cells or following any NIH numbers is thin
The cell of born of the same parents system：BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ES06,
TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14.
2. method described in claim 1, wherein the step 2) includes：The cell that will be obtained from step 1) is transferred to containing into fibre
Tie up the original intestinal tube induction of the micromolecular inhibitor of Porcine HGF (FGF) and transforming growth factor (TGF-b) signal path
It is cultivated in culture medium, so as to obtain original intestinal tube cell.
3. the method described in claim 1 or 2, wherein the original intestinal tube inducing culture includes Desmocyte growth factor
The micromolecular inhibitor SB431542 of son -7 and transforming growth factor signal path.
4. the method described in claim 1 or 2, wherein the pluripotent stem cell is the pluripotent stem cell of induced synthesis.
5. method described in claim 1, wherein the efficiency for generating the hepatoblast of coexpression HNF4A and PROX1 is more than 60%.
6. method according to claim 1 or 2, wherein the pluripotent stem cell is mammalian cell.
7. method according to claim 1 or 2, wherein the pluripotent stem cell is mouse or people's cell.
8. method according to claim 1 or 2, wherein the pluripotent stem cell is people's cell.
9. the hepatic parenchymal cells that the method according to any one of claim 1-8 obtains.
10. the hepatic parenchymal cells described in claim 9, the cell Albumin Secretion, secreted albumin level reach people
The level of primary adult hepatic parenchymal cells Albumin Secretion.
11. the hepatic parenchymal cells described in claim 10, the cell also has selected from storage glycogen, generates Cytochrome P450
Enzyme carries out a phase, phase II metabolic, one or more functions in three-phase transhipment.
12. the hepatic parenchymal cells any one of claim 9-11 is in drug metabolism study, the toxotest or medicine of drug
Application in object screening.
13. identification with metabolic function hepatic parenchymal cells method, it is characterised in that the described method includes detection PROX1 and
The step of expression of the HNF6 in hepatic parenchymal cells.
Applications of the 14.PROX1 and HNF6 in the kit for identifying the hepatic parenchymal cells with metabolic function is prepared.
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| CN105296418B (en) * | 2014-08-04 | 2019-01-18 | 上海赛立维生物科技有限公司 | A kind of method and its application of long-term in vitro culture and amplification liver cell |
| JP2017534269A (en) * | 2014-10-08 | 2017-11-24 | エージェンシー フォー サイエンス，テクノロジー アンド リサーチ | How to differentiate stem cells into liver cell lineage |
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