CN101356264A - Isolated liver stem cells - Google Patents

Isolated liver stem cells Download PDF

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CN101356264A
CN101356264A CN200680048822.0A CN200680048822A CN101356264A CN 101356264 A CN101356264 A CN 101356264A CN 200680048822 A CN200680048822 A CN 200680048822A CN 101356264 A CN101356264 A CN 101356264A
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cell
liver
stem cell
stem
progenitor
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CN101356264B (en
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艾蒂安·索卡尔
穆斯塔法·纳吉米
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Universite Catholique de Louvain UCL
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Universite Catholique de Louvain UCL
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Abstract

The present invention relates to isolated liver progenitor stem cells, and cell population thereof, wherein said progenitor stem cells originate from adult liver, esp. of human. The present invention also relates to the use of said isolated progenitor stem cells in medicine, hepatology, inborn errors of liver metabolism, transplantation, infectious diseases, liver failure. The present invention also relates to methods of isolating these cells, their culture, characterization before and after differentiation, and their use for transplantation, animal models of human disease, toxicology and pharmacology.

Description

Isolating liver stem cells
Technical field
The present invention relates to isolating hepatic progenitor cell or stem cell from adult hepatic, and the purposes in medical science, hepatopathy, liver metabolism inborn defect, transplanting, communicable disease, liver failure.The invention still further relates to method, its culture, the differentiation front and back of separating these cells characterizes and the purposes in animal model, artificial organ device, toxicology and the pharmacology of transplanting, human diseases.
Background technology
Liver is vitals, and it brings into play a lot of important function, synthesizes as the homeostasis of glucose, (xenobiotic) detoxification or the macromole of xenobiotic.Therefore, one of multiple liver function is impaired can produce remarkably influenced to health.According to The World Health Organization (WHO), the sickness rate of global urgency or chronic hepatopathy makes these diseases become the 5th to the 9th the cause of death.So far, to late period hepatic diseases the radical cure method have only hepatic transplantation.The patient's of experience surgery liver transplantation final result is fine, rehabilitates more than 95%.Yet even the new surgical technology that comprises liver segmentation and Living Donor Liver Transplantation donor is arranged, the in short supply day by day of organ makes the mortality ratio in the treatment list that clamps on constantly soaring.Therefore, the important goal in transplantation medicine research proves the potential use of liver cell (liver cell) in liver regeneration and liver disease exactly.
(liver cell transplantation is a kind of emerging method LCT), relates to infusion liver cell suspension in acceptor hepatic portal system in the liver cell transplanting.Its purpose is to recover acceptor liver function and the ill liver parenchyma (liverparenchyma) of reconstruction (repopulation) as transplanting as a result.LCT at first is proved in animal model effectively, has shown that wherein homology liver cell (hepatocyte) can long-term surviving and correct various enzyme defects (summary is referring to Najimi and Sokal.2005.Minerva Pediatr 57 (5): 243-57).
In the middle of the mankind, early stage research is specifically designed to the depletion of treatment acute hepatic.These researchs impel clinical position person is used for other indications with the LCT expansion, and so far, at least 30 cases (people 1997.Transplant Proc 29 (4) such as Strom: 2103-6) of various defectives has been reported in the whole world.In the specific field of metabolic trouble, reported in 13 cases that liver cell is used for the treatment of the purposes of I type Crigler-Najjar syndrome, ornithine cycle defective or orphan disease such as baby Refsum disease.These studies have shown that the implantation of liver cell in liver parenchyma and the improvement that reaches 18 months status of patient subsequently after transplanting.
Yet, because the mature human's liver cell that is used to transplant is still limited, in fact more or less the same limited with full liver operability, research also is intended to obtain transplantable cell from other sources, as progenitor cell and stem cell from embryo or adult source, these cells for example can be at amplification in vitro, and can especially be divided into the ripe liver cell of function after transplanting in the body.Therefore, press for the useful method in treating the relevant various diseases of liver related disease or the patient's condition that exploitation makes new advances, when especially considering the available treatment deficiency of present these diseases of great majority.
In history, do (ES) cell and infinitely clone the ability that division and multipotency are divided into the daughter cell (daughter cell) of whole tissue owing to observe the embryo, it once was considered to only participate in organ and took place.On the other hand, the regenerative process in the adult organ is in typical case owing to adult progenitor cells.Yet, consider the stem cell of in the adult organ, having found to express embryo's mark, this theory is corrected.When therefore, describing stem cell and progenitor cell feature now not only based on growth course (embryo is to adult) also based on the existence of specific cell mark.In fact, the expression of cell sign thing such as membranin or transcription factor can great changes have taken place along with differentiation pathway, and they have reflected various stimulations (as environmental stimulus) and cell requirements.Usually in atomization, observe stem cell and will stop to show mark such as the Oct-4 that indicates its versatility gradually, help the mark of follow-up phase such as the mark of particular lineage and express.As the example of an indefiniteness, Oct-4 loses gradually along with maturation, and the cell that enters the entoderm pedigree on the other hand begins to express alpha-fetoprotein.
With regard to carry out liver regeneration by Transplanted cells with regard to, can consider multiple possible derived cell type.For example because the versatility of ES cell, expect its any organ of can regenerating.In fact, this road is extensively explored in this area.Yet the ES cell tends to cause tumor growth when the ES cell is introduced in any other tissue in addition of uterus (inutero).Therefore, use because carcinogenic risk still is restricted in its body.Even if the ES cell is in the vitro differentiation success before, be used for the human safety inadequately of inoculating all.
Safer alternative is to use adult progenitor cells, and unlike the ES cell, it tends to show the daughter cell of limited clone's splitting ability and the more limited destiny of differentiation generation.In the liver, adult progenitor cells such as elliptocyte (bile duct cell and liver cell precursor) or microhepatia cell like cell had been described.Yet its rareness in normal adult organ makes its medical usage become difficult.
Therefore, have and reduce or do not depart from the major progress that to represent the Transplanted cells source to carcinogenic risk and the adult stem cell that demonstrates clone's splitting ability.Polytype adult stem cell is transplanted in the research at liver cell at present and is estimated.For example, be divided into the more ability of mature cell owing to have from another pedigree commentaries on classics, (mesenchymal stem cell MSC) studies to the mescenchymal stem cell from periphery or Cord blood.And, also aspect the liver regeneration potential myeloid hemopoietic stem cell is being studied.
The vitro characterization of adult stem cell is difficulty still, the present acceptable in this area is the mark that this sign can preferentially comprise detection (i) embryo origin or pedigree (especially mesoderm, entoderm, ectoderm or hemopoietic stem cell), (ii) reflect the expression of the mark of level of differentiation, thereby possible different offsprings have been indicated to a certain extent, destiny in the external or body after (iii) breaking up.Therefore, characterize and distinguish the adult stem cell that obtains from normal liver and can comprise easily whether evaluation exists following mark: (i) reflect the mark of the complicated embryo's origin of this organ, (ii) break up the mark (as there being albumin) and the (iii) mark of at least one indication stem cell destiny.
According to present knowledge, liver mainly is derived from entoderm, and liver cell is the part of entoderm pedigree (lineage).Yet hepatocellular formation also relates to the interaction between entodermal epithelium and the cardiogenic mesoderm.In addition, in fetation, hematopoietic cell generates and occurs in the liver.Consider developmental this influencing each other, need open thinking during the mark that in mentioning the adult hepatic stem cell, exists, because the mark of entoderm, mesoderm and/or hematopoiesis system all can be expected.
When assessment level of differentiation and cell type ownership, can estimate different cell sign things, as what carry out subsequently in the disclosure.For example, some mark reduces or disappears in cell differentiation procedure, and other marks can increase or occur, and also has some can be maintained to specialization and functional cell.According to limiting examples, between the organogenetic period, promptly between fetus period, think that liver parent cell (hepatoblast) is the common progenitor cell that forms essence (especially liver cell and courage cell), its express cell Keratin sulfate-7 (cytokeratin-7; CK-7) and CK-19, albumin and alpha-fetoprotein.In the adult hepatic, the known common progenitor cell of liver cell and courage cell is elliptocyte (oval cell), and it expresses CK-19, albumin and alpha-fetoprotein.After being divided into the courage cell, CK-19 and albumin continue to express, and the expression of CK-7 then is tending towards stopping (thinking a feature of more immature cell).On the other hand, liver cell is kept alpha-fetoprotein and albuminous expression, but does not express above-mentioned CK.Remain this example, the sign that this shows stem cell is complicated, but preferentially the service marking thing estimates indicator cells type or characteristic.
According to the inventor's understanding, isolate progenitor cell from normal adult hepatic has been described in former research, and described cell has represented more than a kind of cell fate.The adult hepatic stem cell that can also be divided into liver cell (and preferably only having liver cell destiny) at in-vitro multiplication was not in vivo still described.In addition, complicated technology is used in former research, separates liver stem cells as FACS, calcic medium or specific density gradient.
Therefore, a target of the present invention just provides progenitor cell or the stem cell with the new adult hepatic source of improving characteristic, and especially can be used for for example liver cell transplanting.The present invention also provides the simple method of separating described cell.
Summary of the invention
The invention provides from progenitor cell or stem cell, the clone that comprises this progenitor cell or stem cell or the cell mass in the adult hepatic source that normal liver tissue obtains.Method, its culture, the differentiation front and back of separating these cells characterize, and the purposes in transplanting, animal model of human disease, toxicology and pharmacology is also within the scope of the invention.
On the one hand, the present invention has realized a kind of novel isolating progenitor cell or stem cell (vertebrates, preferred mammal, more preferably human cell), it is derived from adult hepatic, be characterised in that coexpression (promptly being positive) liver cell mark albumin (ALB) and one or more plant other livers or liver cell mark possibly to following, preferred CD29, alpha-fetoprotein (AFP), a kind of in α-1 antitrypsin and/or the MRP2 translocator, more than a kind of or whole marks, and at least a mesenchyme mark, especially mark CD90, CD44, CD73, a kind of in vimentin (vimentin) and the α-smooth muscle actin (ASMA), more than a kind of as 2,3 or 4 kind or all.Described adult hepatic progenitor cell or stem cell can also be expressed as follows in the molecule of indication liver cell sample feature or function a kind of, more than a kind of or whole: G6P, CYP1B1, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2.The feature of described adult hepatic progenitor cell or stem cell can also be following a kind of, more than a kind of or all marks: hematopoiesis mark CD45 and CD34 are negative at least, also can one or more other hematopoiesis marks, for example, CD105, HLA-DR are negative, bile duct cell epithelium mark cytokeratin-19 (CK-19) and can being negative by more epithelium marks; At least undifferentiated stem cell markers CD117 and Oct-4 are negative, and can be a kind of or more than a kind of embryonic stem cell mark; The low expression level of AFP.Preferably, described adult hepatic progenitor cell or stem cell have mesenchyme sample form, especially comprise monolayer growth, flat pattern, significantly tenuigenin and/or have in the ovogonium nuclear of one or two kernel a kind of, more than a kind of or all forms.
In one embodiment, the present invention provides isolating stem cell from adult hepatic particularly, and it presents CD90, CD29 and the CD44 positive, the albumin positive, the vimentin positive and α-smooth muscle actin positive.In one embodiment, isolating stem cell also presents CK-19 feminine gender, CD45 feminine gender, CD34 feminine gender and CD117 feminine gender.The present invention also provides the cell mass that comprises the progenitor cell mescenchymal stem cell with at least three following features: the detectable albumin of antibody is expressed; The expression of the detectable vimentin of antibody; The detectable α of antibody-smooth muscle actin is expressed; CK-19 disappearance, CD45 disappearance, CD45 mark disappearance, CD34 mark disappearance, CD117 mark lack, have the CD90 mark, have the CD29 mark or have the CD44 mark.Preferred cell has all above-mentioned features.In a preferred embodiment, stem cell is people's liver stem cells.
In one embodiment, the isolating stem cell that comes from adult hepatic presents CD90, CD73, CD29 and the CD44 positive and the albumin positive, the vimentin positive and α-smooth muscle actin positive.
The present invention also provides a kind of acquisition to comprise the method for isolating progenitor cell or stem cell or the cell mass of described progenitor cell or stem cell, this method comprises: adult hepatic or its part of (a) dissociating is so that form the primary cell group from described adult hepatic or its part, (b) with primary cell group bed board above substrate, make cell attachment on it, and (c) from primary cell group culturing cell, cell attachment in described substrate at least 7 days, preferably at least 10 days, have 13 days at least, or at least 15 days.
The present invention also provides a kind of method that obtains isolating liver stem cells or its cell mass, wherein may further comprise the steps: from the adult hepatic culturing cell according to the inventive method, isolating hepatocytes therefrom, bed board liver cell and cultivated described liver cell at least 7 days, preferably at least 10 days, at least 13 days, or at least 15 days.
On the other hand, the invention provides the isolating adult hepatic progenitor cell or the stem cell of using the obtainable or direct acquisition of method of the present invention, clone and/or the cell mass that comprises described adult hepatic progenitor cell or stem cell.
On the other hand, the inventor has set up specific adult people hepatic progenitor cell or stem cell cell mass (clone) according to the present invention, and on February 20th, 2006 described isolated cells system is preserved in Belgian microorganism cooperation preservation center (Belgian Coordinated Collections of Microorganisms (BCCM/LMBP)) according to budapest treaty (Budapest Treaty), preserving number is that LMBP 6452CB (is authorized by international preservation mechanism; Identify with reference to providing: ADHLSC) by the preservation people.Therefore, the present invention relates to be preserved in the BCCM preserving number is LMBP 6452CB isolated cells, clone and the cell mass of (being called " LMBP 6452CB " clone herein), its subbreed comprises clone's subbreed, also relate to its offspring, comprising breaking up the offspring, especially liver cell or liver cell like cell prepared therefrom also relate to the derivative of its genetic modification.
The present invention also provides the composition that comprises according to the present invention isolating hepatic progenitor cell or stem cell or its cell mass.Preferably, liver cell is a human hepatocytes, or mammiferous liver cell.
Have several important advantages according to progenitor cell of the present invention or stem cell (what will say especially is to include but not limited to LMBP6452CB system).For example, unlike the cell of embryo origin, this progenitor cell or stem cell source be in adult, and can show risk lower not controlled (tumour) when being used for the treatment of and increase or vicious transformation.
In addition, the contriver finds, obviously do not show according to progenitor cell of the present invention or stem cell and (for example to be divided into the mesoblastema type, bone or chondrocyte, phoirocyte) ability, the dystopy that reduces this tissue when using this cell or cell implanted liver organization forms (ectopic formation).
The inventor finds that also progenitor cell of the present invention or stem cell can especially preferentially be divided into liver cell or liver cell like cell, and this just makes them be particularly suitable for reconstruct in liver (reconstitute) hepatocyte function.
The liver derived stem cell that progenitor cell of the present invention or stem cell were obviously described before being different from aspect for example morphological specificity and the marker expression is as elliptocyte.
Especially useful in medical science, hepatopathy, liver metabolism inborn defect, transplanting, communicable disease, liver failure according to adult hepatic progenitor cell of the present invention or stem cell.According to hepatic progenitor cell of the present invention or stem cell are especially transplanted at (people) liver cell, preparation people liver cell is transplanted animal model, biology-artificial liver, external liver cell system with suffer from human liver disease's animal model, liver metabolism filler test (pharmacokinetics, cytotoxicity, genetoxic) and useful in the gene therapy instructed of liver cell.Can further be divided into liver cell according to hepatic progenitor cell of the present invention or stem cell.
The present invention also provides and comprises according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the randomly offspring of genetic modification) pharmaceutical composition and pharmaceutically acceptable carrier.Preferably, described liver cell is the human liver cell, or the mammalian liver cell.
The present invention also is provided for treating the method for hepatic diseases, comprising use significant quantity according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the randomly offspring of genetic modification).In one embodiment, hepatic diseases, include but not limited to pku and other amino acid metabolism diseases (aminoacidopathies), hemophilia and other thrombin defectives, familial hypercholesterolemia and other lipid metabolism disorders, urea cycle disorder, glycogenosis (glycogenosis), galactosemia, fructosemia (fructosemia), tyrosinemia (tyrosinemia), protein and carbohydrate metabolism defective, organic aciduria, mitochondrial disease, peroxysome and lysosome are not normal, protein synthesis is unusual, liver cell translocator defective, glycosylation defect, hepatitis, liver cirrhosis, congenitalDy olism, acute hepatic failure, acute liver infects, acute chemical toxicity, chronic liver failure, cholangitis, cholehepatocirrhosis, Alagille syndrome, alpha-1-amtitrypsin deficiency, autoimmune hepatitis, Biliary atresia, liver cancer, the hepatic pouch venereal disease becomes, fatty liver, galactosemia, gallbladdergallstonecholetithiasis, Gilbert syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, and other virus infection type hepatitis, porphyria, primary sclerosing cholangitis, the Reye Cotard, sarcoidosis, tyrosinemia, I type glycogen storage disease (type I glycogen storage disease), or Wilson's disease (Wilson ' s disease).
The present invention also is provided for therapeutic gene and expresses wrong method, this method comprises: (i) the functional copy of a gene is introduced the group who is used to provide conversion according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (offspring who wherein comprises differentiation, especially liver cell or liver cell like cell); The group that (ii) near small part transforms introduces patient's liver, and this patient is just needing the functional copy of this gene.As an alternative, the group who transforms can be introduced inhuman mammiferous liver, so that produce the animal model of a new hepatic pathology.
The present invention also is provided for therapeutic gene and expresses wrong composition, described composition comprises that according to the present invention the hepatic progenitor cell of introducing the conversion that the gene function copy is arranged or stem cell, its clone or cell mass or its offspring are (comprising breaking up the offspring, especially liver cell or liver cell like cell, the randomly offspring of genetic modification).
The present invention also is provided for therapeutic gene and expresses wrong pharmaceutical composition, described pharmaceutical composition comprises according to the present invention introduces hepatic progenitor cell or stem cell, its clone or cell mass or its offspring (offspring who wherein comprises differentiation that the gene function copy is arranged, especially liver cell or liver cell like cell), and pharmaceutically acceptable carrier.
The present invention also is provided for strengthening the method for impaired or ill liver regeneration, described method comprises: to liver use significant quantity according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the randomly offspring of genetic modification).
The present invention also provides the liver supplementary unit that comprises pedestal (housing), described pedestal holds according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the randomly offspring of genetic modification).
The present invention also is provided at external method of carrying out toxotest, described method comprises to be made according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the offspring of genetic modification randomly) Contact test reagent, and observe (if any words) at least a test agent is to the effect of liver cell population.Preferably, at least a effect is comprising the effect of pair cell vigor, cell function or this two aspect.
The present invention also is provided at external method of carrying out drug metabolism study, described method comprises: (i) make according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring, especially liver cell or liver cell like cell, the offspring of genetic modification randomly) Contact test reagent, and (ii) observe (if any words) at least aly relate to the variation of test agent after the predetermined testing period.Preferably, at least a variation, comprising test agent in the variation aspect structure, concentration or this two.
The present invention also provides and infects the method that effective agents is tested to being used for the treatment of liver, described method comprises: (i) go to infect according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring with the target infectious agent, especially liver cell or liver cell like cell, the randomly offspring of genetic modification) in order to infected group to be provided; (ii) make the test agent that is subjected to infectious group contact predetermined amount, and (iii) observe (if any words) contact is to being infected group's effect.In one embodiment, infectious agent comprises a kind of microorganism.In another embodiment, infectious agent comprises one or more kind viruses, bacterium, fungi or its combination.In a specific embodiments, the effect of observation comprises the former effect to virus replication of viral communication, and preferably, the virus infection agent comprises hepatitis virus.
The present invention also provides a kind of productive target method of protein, described method comprises: the functioning gene of the target protein of (i) will encoding is introduced according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (offspring who wherein comprises differentiation, especially liver cell or liver cell like cell), (ii) be fit to transcribe, translate and condition that randomly posttranslational modification takes place under hatch described cell mass, and (iii) gather in the crops target protein.Preferably, liver cell is the human liver cell.In one embodiment, target protein comprises vaccine antigen.
The present invention also is provided at the method for in the external or body growth and the liver cell differentiation of liver being studied, and described method comprises: making in external or body according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up the offspring) contact influences the condition of differentiation condition and observes at least a pair cell group's effect.
The method that the present invention includes cell, its preparation, sign, cultivation and produce.
The present invention comprises that also the culture by these cells of cryopreservation comes these cells of preservation.
The present invention also comprises the technology of Transplanted cells among the animal and the mankind.
The present invention comprises that also liver stem cells according to the present invention prepares the purposes of the medicine that is used for the treatment of above-mentioned disease.The present invention also comprises the purposes of liver stem cells according to the present invention in test kit or part test kit.
Description of drawings
Fig. 1 represents that the present invention comes from the progenitor cell of human adult liver or the form outward appearance (ADHLSC) of stem cell, and described cell such as embodiment 1 preparation are observed through using opticmicroscope (differing) after 1 month the cultivation.A, lower converging (confluence); B, higher converges.Magnification is 100 *.
Fig. 2 A: present the progenitor cell that comes from people's adult hepatic according to the present invention or the immunofluorescence dyeing of stem cell (ADHLSC), as prepared among the embodiment 1, after cultivating in 1 month, α-smooth muscle actin (A1), vimentin (A2) and albumin (A3, polyclone, A4, mono-clonal).B: and human liver cell (hHep, 2 roads), human astrovirus cell (hSC, 3 roads) and human hepatoblastoma (HepG2,4 roads) are compared, the RT-PCR gene expression profile in the described clone (ADHLSC, 1 road).
Fig. 3 represents that progenitor cell or stem cell (ADHLSC) that the present invention comes from people's adult hepatic become liver cell sample pedigree in vitro differentiation.Cell breaks up as described in embodiment 1, obtains image at the 2nd day (J2) of process, the 14th day (J14) and the 30th day (J30).
Fig. 4 represents as describing in detail among the embodiment 1, to show that these cells become the liver cell of differentiation with several phenodins in the chimeric liver of the uPA/SCID mouse of undifferentiated ADHLSC Transplanted cells and eosin colored graph picture.
Fig. 5 represents several the painted images of human albumin, shows in embodiment 1, transplants the 10 week observed liver cell sample groups' in back humanized with undifferentiated ADHLSC.
Detailed Description Of The Invention
As used herein, unless context has clearly indication in addition, otherwise there is not the object of quantifier restriction to anticipate Think to comprise one and a plurality of referent. As an example, " cell " refers to one or more Cell.
As used herein, term " comprises ", " comprising " and " containing " be synonym, is to have bag Capacitive or open, and do not get rid of the extra member, key element or the method step that do not describe in detail.
All lists of references of quoting in this specification are all incorporated this paper by reference into. Especially this paper The instruction of all references that relate to is specially incorporated into by reference.
Obtain cell of the present invention
On the one hand, the invention provides a kind of CFU-GM that obtains to separate or stem cell or comprise described ancestral The method of the cell mass of cell or stem cell, described method comprises: adult hepatic or one (a) dissociate Part is in order to form the primary cell group from described adult hepatic or its part, (b) with primary cell Group's bed board adheres on it cell above substrate, and (c) from primary cell group cultured cell, Cell is attached in the described substrate at least 7 days, and preferably at least 10 days, have 13 days at least, or at least 15 days.
As used herein, term " cell of separation " typically refer to not with one or more cells or The cell of one or more cell component combinations, and they are combinations in vivo. For example, separation Cell can leave its natural surroundings, or can be from the propagation of the cell that leaves natural surroundings, For example, vitro proliferation.
As used herein, term " external " refers to outside animal or human's body or the outside. Used herein Term " external " is understood to include " exsomatizing ". Term " exsomatize " typically refer to leave animal or The tissue of human body or cell and in external preservation or propagation, as in culture vessel.
Term " cell mass " typically refers to one group of cell. Unless otherwise, otherwise this term refer to The cell that is separated by this paper forms or comprises the cell colony of the cell of separation herein.
Cell mass can form and maybe can comprise at least part of common phenotype that has by having altogether isophenic cell Cell. When cell basic simlarity or when consistent on one or more notable features, think the cell tool Common phenotype is arranged, its feature include but not limited to form outward appearance, certain cell component or product (as RNA, Protein or other materials) the having or not or level, the activity of certain biochemical route, multiplication capacity of expression And/or dynamics, differentiation potential and/or to the response of differentiation signal or in vitro culture behavior (as, attached , non-cohesive, monolayer growth, growth kinetics etc.). Therefore this notable feature can define one Individual cell mass or its part.
When claiming that herein cell mass is " heterogeneity " (heterogeneous) time, this ordinary representation cell mass Comprise two or more and do not have altogether isophenic cell or cell part, comprise two kinds such as cell mass Or the cell of more kinds of different cell types. Such as but not limited to, the heterogeneity cell mass can separate certainly Liver, and can comprise multiple liver cell type includes but not limited to that liver cell is (as big with little Liver cell), bile duct cell, Kupffer cell, HSCs (Ito cell) and liver Endothelial cell.
When claiming that herein cell mass is " homogeneous " (homogeneous) time, it is altogether isophenic by having Cell forms. The most cells that comprise when claiming cell mass " basic homogeneous " herein have common table Type. " basic homogeneous " cell mass can comprise at least 70%, such as at least 80%, and preferably at least 90%, As at least 95% or even at least 99% cell have common phenotype, as the phenotype specifically mentioned (as The phenotype of CFU-GM or stem cell). As used herein, therefore term used herein " basic homogeneous " The group that also can comprise homogeneous.
Term " cell mass that comprises CFU-GM or stem cell " relates to as defined here and comprising at least A kind of CFU-GM or stem cell, in typical case as defined here part CFU-GM or stem cell Cell mass. Usually, the CFU-GM of described part or stem cell can have common phenotype.
Term " CFU-GM " typically refer to do not have specialization or a relatively less specialization and proliferation potential arranged Cell, this cell or its offspring can produce at least a relatively more cell type of specialization. Such as but not limited to, CFU-GM can produce along the offspring of one or more pedigree differentiation and produce relatively The cell of specialization more and more, wherein this offspring and/or relatively more and more the cell of specialization can self just CFU-GM, or even produce the eventually cell of end differentiation, that is: the complete cell of specialization, this can be After the mitosis. Term also comprises the stem cell of definition herein.
When thinking the another kind of relatively more cell of specialization of CFU-GM " generation ", such as but not limited to, CFU-GM is at first through cell division and be not divided into another kind of cell, perhaps another kind of cell be through CFU-GM or its offspring one take turns or more wheels cell division and/or differentiation after and produce.
The CFU-GM that term " stem cell " refers to can self (namely do not break up and can breed), Wherein the offspring of stem cell or at least its part substantially kept the parental generation stem cell not specialization or phase Phenotype, differentiation potential and multiplication capacity to less specialization. This term comprises can be essentially no The stem cell of limit self that is: is compared with parental cell, and offspring or its part are further bred Ability does not significantly reduce, and the stem cell that shows limited self, that is: and mother cell Compare, offspring or its part further ability of propagation significantly reduce.
Those skilled in the art will know that above-mentioned feature typically refers to CFU-GM and stem cell row in vivo For, fully or at least in part repeat out external and/or stripped under suitable condition.
Based on the ability that produces different cell types, it is complete that CFU-GM or stem cell can be described to usually Energy (totipotent), multipotency (pluripotent), special energy (multipotent) or monoenergetic (unipotent). Single " all-round " cell is defined as can grow (that is: growing) and becomes whole life Object. " multipotency " cell can not grow up to whole organism, comes from all three embryos but can produce The layer cell type, namely in, in and ectoderm. And can produce all cells class of organism Type. " specially can " cell can produce from two or more Different Organs of organism or tissue extremely Few a kind of cell type, wherein said cell type can be derived from identical or different germinal layer (germ But can not produce all cells type of organism layer). " monoenergetic " cell can only be divided into one The cell of clone.
Term " differentiation " or its derivative are used for referring to a kind of process herein, not specialization or relatively The cell of few specialization is by this process relatively more specialization that becomes. In cell individual takes place, describe Word " differentiation " is a relative terms. Therefore, " cell of differentiation " than the cell of comparing and Speech is the cell that a kind of growth enters specific development pathway. The cell of differentiation for example can be that the end is divided eventually The cell of changing, that is: the complete specialization of performance specialization function thin in the various tissues of organism and organ Born of the same parents, but need not be after the mitosis. In another example, the cell of differentiation also can be CFU-GM in the differentiation pedigree, it can further be bred and/or break up. Similar, if the cell ratio The cell of comparing development has entered specific development pathway, and then cell is " specialization relatively more ", wherein the latter to be construed to be " not specialization " or " relatively less specialization ". Relatively more The cell of specialization can be different from aspect the one or more remarkable phenotypic characteristics specialization not or relatively The cell of less specialization, described phenotypic characteristic as, for example, certain cell component or product (as RNA, Protein or other materials) expression have or not or the activity of level, certain biochemical route, form outward appearance, Multiplication capacity and/or dynamics, differentiation potential and/or to response of differentiation signal etc., wherein these spies Levy show specialization relatively more cell further along the process of described development pathway.
The differentiation limiting examples can comprise, as, myeloid-lymphoid stem cell becomes the special energy of specified type The variation of CFU-GM or stem cell, special energy CFU-GM or stem cell become the monoenergetic CFU-GM of specified type Or the variation of stem cell or monoenergetic CFU-GM or stem cell become in the designated cell system the thin of specialization more Born of the same parents' type or the eventually variation of the cell of last specialization. Can be by having the outward appearance of medium specialization degree cell Distinguish Cell Differentiation specialization or less specialization and the cell of specialization more.
Liver organization dissociates
As described, method of the present invention comprises that dissociate adult hepatic or its part are with from described adult liver Dirty or its a part of formation primary cell group's step.
Term " liver " refers to liver organ. Term " liver part " typically refers to liver organ Any part, for the zone of the part of described liver organ or liver origin on amount without any limit System. Preferably, all cells type that exists in the liver organ also can occur in described liver part. The amount of part liver is deferred to actual Consideration on can be at least part of, as obtaining to be enough to implement the present invention Method former generation liver cell needs. This consideration of instruction according to the present invention is for art technology Personnel are apparent. Therefore, such as but not limited to, the part liver can represent (typical case Ground w/w) at least at least 1% or at least 10% or at least of 0.1% or liver organ of liver 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70 % or at least 80% at least 90% or more than. In other limiting examples, the part liver Can be at least 1g or at least 10g at least 100g or at least 200g or at least 300g, At least 400g or at least 500g at least 600g or at least 700g or at least 800g or At least 900g or at least 1000g at least 1100g or at least 1200g or at least 1300g, At least 1400g or more than. For example, the part liver can be lobe of the liver, for example, and lobus dexter or lobus sinister, Or liver split from the operation in the excision the IV part.
Term used herein " adult hepatic " refers to obtain basic fully-developed tissue (tissue Organization) and cell form.
Specifically, the known liver of those skilled in the art experience growth change in a period of time after birth Change the tissue that liver acquisition is mature on the whole during this. For example, in the human subjects, connatae liver Contain a considerable amount of hematopoietic cells, its 1-2 after approximately being born disappears from liver in week substantially. And people's liver contains a group hepatic progenitor cell at birth, and it is basic in some months after birth Replaced by mature hepatocytes and courage cell.
Therefore, in human subjects, " adult hepatic " referred in the rear any time of birth, and be preferably full-time Phase (full term), can be the birth after at least one monthly age, such as at least 2 months, at least 3 months, Such as at least 4 months, at least 5 months, as being born at least rear 6 monthly ages, as, for example 1 year or with Upper, 5 years or above, at least 10 years or above, 15 years or above, 20 years or above or birth The liver of rear 25 years or above object. Therefore, can in human subjects, find " adult hepatic " or Ripe liver, wherein human subjects will be under different situations according to conventional term " baby ", " children ", " youth ", " teenager " or " adult " describe.
One skilled in the art will appreciate that liver is passable in the postpartum different time in the different animals species Substantially reach maturity, can proper interpretation term " adult hepatic " for each species.
Liver or its part obtain from " object ", " donor object " or " donor ", and be wherein " right Resemble ", " donor object " or " donor " can exchange when relating to vertebrate, preferred mammal, Be more preferably the people.
Term " mammal " comprises the animal of any classification, described classification as, include but not limited to, The mankind, domestic animal and farm-animals, zoo animal, physical culture animal (sport animal), pet move Thing, companion animals and animal used as test, wherein animal used as test as, for example, mouse, rat, rabbit, dog, Cat, ox, horse, pig and primate, for example monkey and anthropoid cape.
In an especially preferred embodiment, adult hepatic or its part are from human subjects. As Describe in detail in this specification, according to the present invention from CFU-GM or the stem cell of the liver of human subjects Or clone or its offspring can be advantageously used in, as, especially suffer from the human patients of hepatopathy Research and treatment.
In another embodiment, adult hepatic or its part can be from inhuman animal targets, and be excellent Select the non-human mammal object. According to the present invention from the ancestral of non-human animal or non-human mammal object Cell or stem cell or clone or its offspring can be advantageously used in, as, in identical, phase with it Research and treatment hepatopathy among the species member of pass or other non-human animal or non-human mammal object, Or even the treatment suffer from hepatopathy (such as xenotransplant, comprise non-human animal or non-human mammal The bioartificial liver devices of cell) human patients. Such as but not limited to, especially be suitable for the mankind The non-human mammal cell for the treatment of can be from pig.
The donor object can be that what to live also can be dead, determined according to the principle that this area is accepted, as, for example, " heart-lung " standard (comprising circulation and the irreversible of respiratory function usually stops) or " brain death " standard (comprise full brain usually, comprise brain stem, the irreversible of all functions stops).Obtain and can comprise process known in the art, for example, examination of living tissue, excision or surgical blanking.
Those skilled in the art will know that from least some aspects of donor object acquisition liver or its part and can suffer considering from law and code of ethics respectively.For example but do not limit, obtaining liver organization from the human donor of living can need to satisfy and keep donor survival from now on.Therefore, can have only the part of liver from the human donor of living, to separate in typical case,, thereby in donor, keep enough physiology liver function levels as use examination of living tissue or excision.On the other hand, obtain liver or its part possibility from the non-human animal, but do not need to satisfy non-human animal's survival from now on.For example, the non-human animal just can have been put to death by humanity after obtaining tissue.These and similarly consideration be conspicuous for those skilled in the art, it has reflected law and moral standards and has not related to essence of the present invention substantially.
In one embodiment, liver or its part can obtain from the especially human donor of donor, and described human donor has lasting circulation such as dancing heart, lung or the Spirophore of lasting respiratory function as breathing.Consider morals and Legal Norm, donor can need or not need brain death (may be unsuitable for the survival from now on of human donor as this, but can allow to separate whole liver or its part in the mankind of brain death).Obtain liver or its part has superiority from this donor, do not suffer substantive anoxic (lacking oxygen) because organize, substantive anoxic is normally caused by ischemic (ischemia) (circulation stops).
In another embodiment, as the pleasantly surprised discovery of the inventor, liver or its part can obtain from the especially human donor of donor, described human donor circulates when obtaining tissue and has just stopped, stop to beat and/or respiratory function stops as heart, as the lung of not breathing and the machine that breathes no more.May suffer to a certain degree anoxic, the inventor to find that according to the present invention available progenitor cell or stem cell also can be from these tissues acquisitions although come from the liver of these donors or its part.Liver or its part are obtained within about 24 hours after donor circulation (as heartbeat) stops, as within about 20 hours, as within about 16 hours, more preferably within about 12 hours, within about 8 hours even within more preferably about 6 hours, as within about 5 hours, within about 4 hours or within about 3 hours, more preferably within about 2 hours and most preferably approximately within an hour, stop within about 45,30 or 15 minutes of the back as donor circulation (as heartbeat).
Can be cooled to about room temperature as the above-mentioned tissue that obtains, or subambient temperature, but avoid freezing tissue or its part, especially this freezing meeting to cause nucleus to form or ice-crystal growth usually.For example, can preserve tissue between about 1 ℃ and the room temperature, between about 2 ℃ and the room temperature, between about 3 ℃ and the room temperature or under any temperature between about 4 ℃ and the room temperature, and advantageously be kept at about 4 ℃.As known in the art, organize also and can be kept at " on ice ".Can be at whole or part ischemic stage (that is: the time after the donor internal recycle stops) internal cooling tissue.In other words, tissue can be through the combination of the ischemic that is heated, cold ischemic or hot cold ischemic.The tissue that can will obtain before processing is preserved and is reached 48 hours, preferably is less than 24 hours, as is less than 16 hours, more preferably less than 12 hours, as is less than 10 hours, is less than 6 hours, is less than 3 hours, is less than 2 hours or is less than 1 hour.
Before further handling tissue, the tissue that obtains can be easily, but be not must be stored in as whole or to small part be dipped in suitable matrix and/or can but be not to pour into suitable matrix.Those skilled in the art can select suitable can be before handling the matrix of sustentacular tissue's cell survival.
Method of the present invention comprises that the aforesaid adult liver organization that dissociates forms the primary cell group.
Term used herein " dissociates " and typically refers to the cell tissue structure of disorganize partially or completely or organ, the i.e. partially or completely cell of disorganize or organ and the contact between the cellular constituent.The purpose that it will be appreciated by those skilled in the art that disintegrated tissue or organ is to obtain cell (cell mass) suspension from described tissue or organ.Suspension can comprise independently or individual cells, and physical attachment and two or more cells of forming bunch or group.Preferable separation does not cause or the least possible reduction that causes cell viability.
The method that liver or its part come to obtain from it primary cell group (suspension) that is fit to dissociate can be any method well known in the art, includes but not limited to enzymic digestion, mechanical separation, filtration, centrifugal and combination.Therefore in one embodiment, the dissociate method of liver or its part can comprise that the enzymic digestion liver organization discharges liver cell.In one embodiment, the dissociate method of liver or its part can comprise the physical disturbance of hepatic tissue or separate and discharges liver cell.In one embodiment, the dissociate method of liver or its part can comprise that the enzymic digestion of hepatic tissue and physical disturbance or isolating both combinations discharge liver cell.
The method of above-mentioned dissociate liver or its part is on the books in the art.For example, the method for separating liver cell from liver organization from nineteen sixty for mid-term be known technology just 1967.J Cell Biol 35:675-84 such as () Howard.Utilize the machinery of combination to separate rat hepatocytes, improve (J Cell Biol 43:506-20,1969) through Berry and Friend afterwards with the enzymic digestion technology.This technology through Seglen further develop and become widespread use two the step collagenase perfusion (two-stepcollagenase perfusion) technology (Methods Cell Biol 13:29-83,1976).
Therefore, in one embodiment, dissociate liver or its part come to be or to comprise two step collagenase perfusion technology from its method that obtains primary cell group (suspension).Those skilled in the art will know that because top described technology is open multiple improvement has been described in the present invention and/or can have been imagined, and they are included in the scope of the present invention.
For example but unrestricted, subsequently two step collagenase perfusion technology of routine are summarized.For full liver, intubate (cannulae) can be placed existing main liver vessel, and pass through sutured.For the part or the sections of liver, intubate can place patient vessel's opening of cut surface, and passes through sutured.In this case, need the little openings in blood vessels of sealing to prevent that primer solution is from the cut surface seepage usually.With the free damping fluid perfusion of the divalent cation of 37 ℃ of following preheatings liver organization, described damping fluid contains cation chelating agent, for example, and as ethylenediamine tetraacetic acid (EDTA) (EDTA) or ethylene glycol tetraacetic (EGTA).Damping fluid can comprise salts solution, as, for example, N-2-hydroxyethyl piperazine-N '-ethylsulfonic acid (HEPES), Williams E substratum, Hanks ' balanced salt solution or Earle ' s balanced salt solution can also comprise salt such as sodium-chlor and Repone K etc.This causes cell is maintained together desmosome structural damage.Use the damping fluid perfused tissue then, damping fluid contains divalent cation, as Ca 2+And Mg 2+, and the extracellular matrix degrading enzyme of performance digestion function of organization.In former generation,, liver cell, especially liver cell be released mechanicalness to finish dissociation process through the physical disturbance of gentleness usually, as with comb rake, vibration, with the strainer extruding, as Stainless Steel Filter, cheese cloth (cheesecloth) or nylon fabrics.This strainer has the sieve size (sieve size) that allows liver cell to pass through, for example and unrestricted, and about 0.1 millimeter or above, about 0.25 millimeter or above, about 0.50 millimeter or above, about 1 millimeter or above, about 2,3,4 or 5 millimeters.A series of strainers that can use mesh to reduce gradually make tissue dissociate step by step and discharge cell.With the dissociated cell of damping fluid rinsing, damping fluid contains proteinase inhibitor, serum and/or blood plasma and comes deactivation collagenase and the used enzyme of other filling process, separate through low-speed centrifugal, as (all viable cell can precipitate easily basically between 10 * g and 500 * g, and dead cell and cell debris are removed substantially), come the purifying cells suspension with the throw out that ice-cold damping fluid washing obtains.
Isolating hepatocellular quality and quantity depends on the different and different of the type of the composition of quality as used tissue, perfusion damping fluid and enzyme and concentration.Enzyme commonly used includes, but not limited to collagenase, PRONASE A, trypsinase, Dispase (Dispase), Unidasa, thermolysin (Thermolysin), pancreas enzyme (pancreatin) and combination thereof.Collagenase is the most frequently used, and preparation (as from Clostridium histolyticum) from bacterium usually can be usually be made up of the mixture without the enzyme of fine purifying, and inconsistent enzyme effect wherein can be arranged.Some enzyme shows protease activity, may cause undesirable reaction and influences the quality and the quantity of vigor/healthy cell.Those skilled in the art will know that the enzyme that will use enough purity and quality obtains the alive liver cell group.
The additive method that obtains former generation liver cell can not comprise the enzymic digestion technology.Mechanicalness is broken and is used widely, although often being less than by collagenase digesting, the output of the liver cell of producing by this process obtains, and less consistence.Yet nearest method has been developed and has been had remarkable achievement, and 2002.Cell Biol lnt 26:1003-1006 such as () Kravchenko comprising in the refrigerative environment, combines the perfusion of sucrose-EDTA with controlled vibration.With the sucrose solution situ perfusion liver that contains EDTA (pH7.4).After the perfusion, liver is separated from health, place dish, segment in the ice-cold matrix of the small volume of packing into.By using controlled mechanical vibration depolymerization (the mechanical vibrational disaggregation of homogenizer motor; MVD) mode discharges the cell of liver fragment.The homogenate that this method obtains can provide the initial suspension of liver cell subsequently with coarse net filtering.Cell can be suspended in the matrix, regains by centrifugal.Therefore, in one embodiment, can separate liver cell or its part by mechanical destruction.
The two step collagenase technology that those skilled in the art will know that are particularly useful for discharging liver cell from liver organization at least.The cell suspension that obtains by described technology can comprise the liver cell of considerable part, also can contain other liver cell type.As mentioned, the inventor has been found that this cell suspension is the especially suitable material that is used to obtain progenitor cell of the present invention or stem cell.
In one embodiment, the method of liver or its part of dissociating can form cell suspension (can be optimized by those skilled in the art easily), described suspension comprises at least 10%, as have 20%, at least 30% at least, as at least 40%, at least 50%, as have 60%, at least 70% at least, as have 80% or at least 90% at least, or about 100% independent cell can be arranged at most, promptly unicellular.
As mentioned, thus the liver organization that dissociates provides the primary cell group from described adult hepatic or its part.
As usefulness herein, term " primary cell " comprise from object tissue or organ as the cell of existence in the cell that exists the cell suspension that obtains by dissociate (be bed board before cell mass), the outer planting tissue, when the bed board first two types of fronts cell, from the cell of the cell suspension of the cell of bed board first.Term " subculture cell " is meant the cell in all subsequent steps in the cultivation.Therefore, when the primary cell of bed board goes down to posterity first,, as all cells in follow-up going down to posterity, be referred to herein as the subculture cell so as from substrate surface picking and bed board again.
Definition herein and normally inhomogenous by the primary cell group of liver or its part acquisition of dissociating, that is: can comprise the cell that belongs to cell type in more than one livers.Typical liver-composing type cell type includes but not limited to liver cell, bile duct cell, (bile duct road cell), Kupffer cell, hepatic stellate cell (Ito cell), elliptocyte and liver endothelial cell.Above-mentioned term has this area its meaning and should extensively be interpreted as any cell type that comprises like this classification herein.Liver-composing type cell type also comprises essence and non-essence liver cell.
Still unrestricted as further specifying, " liver cell " comprises epithelium, essence liver cell, the liver cell that includes but not limited to vary in size (as, " little ", " in " and the liver cell of " greatly "), ploidy (as, diploid, tetraploid, octoploid) or other features.For example, some authors proposes, and the liver cell of " greatly " of its definition is the parenchyma of responsible liver physiology function, and the liver cell of " little " growth is provided is that the storehouse of hepatocellular progenitor cell (is seen, as BiochemBiophys Res Commun 214:310-7 such as Mitaka, 1995).Also still unrestricted as further specifying, " bile duct cell " comprises the epithelial cell of bile duct.Also still unrestricted as further specifying, " elliptocyte " comprise particular shape (as, karyomorphism) and the cell sign thing cell of expressing, well-known as this area institute, being considered to is the progenitor cell that can produce liver cell and bile duct cell (.KN.2003.J Gastroenterol Hepatol 18:4-12 such as Lowes under given conditions; 1999.J of Hepatology 31:497-507 such as Yi).
Therefore, in one embodiment, inhomogenous former generation liver cell group can comprise at least two kinds, as at least three or four or the cell type of more kinds of liver-composing types, as belong to all or the cell of all livers-composing type cell type substantially, include but not limited to the cell type of listing above.No matter still external in the body those skilled in the art understand the liver cell type that inhomogenous group describes before can comprising,, and this area liver cell type of not describing as yet, classify, separating and/or characterizing in the past.
Those skilled in the art know that also the heterogeneity cell mass can include but not limited to various liver cell types, and the relative proportion of described various liver cells is same as or is same as substantially the relative proportion that exists in its liver that separates the place or its part.For example, the technician knows, one or more other cell types are compared, and specific liver organization dissociating method can cause one or more cell types of more effective separation, wherein the cell suspension of Huo Deing can be carelessly or targetedly enrichment aforesaid one or more cell types.In addition, some dissociating method can have different influences to the survival and/or the vigor of different liver cell types.The technician also knows and is used for the method for enrichment by this area of the cell mass that separates liver or its part and obtain at one or more target liver cell types.These methods include but not limited to differential centrifugation, buoyant density gradient centrifugation, filtration, cell elutriation (elutriation), affinity purification, protease digestion etc.
In one embodiment, former generation liver cell uneven a group can comprise and belong to all or the cell of all livers-composing type cell type substantially.The method of undocumented hepatic progenitor cell or stem cell type before the inventor finds to be used to obtain.In any case the contriver does not wish that the source of described novel progenitor cell or stem cell is subjected to the restriction of any hypothesis.
Such as but not limited to, described progenitor cell or stem cell or its ancestors are Already in the liver, as liver essence or non-essence.For example, this ancestors can have and isolating progenitor cell or identical, the similar or different phenotype (can change ancestors' phenotype as cultivation according to the present invention) of stem cell.As an alternative, or in addition, isolating progenitor cell or stem cell can be because of changing as differentiation or dedifferente and produce one or more liver cell types, as known or unknown liver cell type in the past.Given this, method of the present invention can be preferably from representing all or the cell of all liver cell types substantially.
The contriver has been found that and can obtain progenitor cell of the present invention or stem cell easily from the cell mass that forms by separation liver or its part that wherein said cell mass comprises liver cell.Therefore, suitable method of separating liver or its part according to the present invention forms and comprises hepatocellular cell mass.Be not bound by any theory, the contriver thinks that hepatocellular mode together discharges to discharge at least for the progenitor cell of invention or stem cell or its ancestors from liver organization (liver obtains by separating) from liver.
In one embodiment, the method of liver or its part of dissociating can form and comprise the hepatocellular cell mass of certain proportion, wherein about at least 10%, about at least 20%, at least 30%, at least 40%, preferably about at least 50%, as at least 60%, more preferably about at least 70%, as about at least 80% in addition more preferably about at least 90% or above, as about at least 95%, about at least 96%, about at least 97%, about at least 98% or about at least 99%.The inventor find to form the liver cell that comprises significant proportion (as, as mentioned above about at least 50% or more than) the separation liver or the method for its part provide suitable initiator cell group to be used to obtain progenitor cell of the present invention or stem cell.
In a preferred embodiment, comprising the hepatocellular cell mass of aforementioned proportion can obtain by dissociate liver or its part, does not wherein comprise further the step at liver cell and/or other cell type enrichment of cell groups.
In another embodiment, can be by dissociate liver or its part and a step or multistep at liver cell and/or other cell types especially liver cell and enrichment of cell group's step obtains to comprise the hepatocellular cell mass of aforementioned proportion.Yet, the technician knows, in method at liver cell, its subgroup or other cell type enrichment of cell groups, when progenitor cell of the present invention or stem cell or its ancestors are being suitable for discharging can discharge from liver under the hepatocellular condition of dissociating the time, can but not always, with liver cell or one or more hepatocellular subgroups (as, the liver cell of " greatly " or " little ") or other cell types altogether-purifying.System of selection is used for especially liver cell of enrichment liver cell and/or other cell types, thereby keeps progenitor cell of the present invention or stem cell or its ancestors in the cell mass that obtains, and this belongs in those skilled in the art's the limit of power.
In addition, those skilled in the art will know that progenitor cell of the present invention or stem cell or its ancestors can have some character (as the expression of physical properties or surface marker) and make it be able to enrichment the cell mass that obtains from liver by suitable separation method.Determine isolating or at progenitor cell of the present invention or stem cell or its ancestors and the cell mass fraction (fraction) of enrichment belongs within those skilled in the art's the limit of power based on one or more principles.This can cultivate cell from various test flow points by the method according to this invention, and determines which flow point produces progenitor cell of the present invention or stem cell is realized.
" group of enrichment " of cell is meant a kind of like this cell mass, its neutralization can be in vivo or the cell of in the cell mass of enrichment, finding compare, one or more cell types exist with bigger relative proportion.
Bed board is from the primary cell of liver organization
Method of the present invention comprises the primary cell group of cultivation by the method acquisition of the liver organization that dissociates as described here.For this reason, the primary cell group of liver cell is plated on makes cell attachment on it in the substrate.
Term used herein " bed board " and inoculation synonym, and typically refer to cell mass introduced and can promote the cell survival introduced and/or the external environment of growth.Usually, described environment can provide in the system that a kind of and surrounding environment suitably distinguish, thereby, can avoid the exchange of substance between described environment and the surrounding environment (therefore to avoid, reveal as the pollution of environment or culture medium or from the cell of culture medium), allow the exchange (as the continuous exchange of switching part or all culture mediumes, gas once in a while or cultivate back harvested cell etc.) of continuous or intermittent other useful matter compositions between described environment and the surrounding environment.Usually, can set up the environment that is suitable for culturing cell in the culture vessel known in the art, as, for example, various forms of cell cultures are shaken bottle, orifice plate and plate.
Among the present invention, cell (as former generation liver cell) is plated on allows the substrate of cell attachment on it, promptly cell attachment or attaching are not repelled in its surface.This can by as cell be plated in the culture systems (as cultivating vessel) implement, wherein said culture systems presents one or more substrate surfaces that is suitable for cell attachment.When the cell suspension (as the suspension in the matrix) of culture systems was introduced in described one or more substrate surface contacts, cell adhesion between cell and the substrate surface can take place.Therefore, term " with cell inoculation in allowing the substrate of cell attachment on it " is meant cell is introduced culture systems, the substrate surface that is characterized as at least a extensive compatible cell adhesion of described system, thereby the cell of inoculation can contact described substrate surface.The rule of keeping adherent cell culture is being known in the art.
Usually, allowing the substrate of cell attachment on it can be any very hydrophilic substrate.As known in the art, culture vessel, shake bottle, orifice plate, plate etc. as cultivation, usually can form by various macromolecular material manufacturings, include but not limited to polyacrylic ester, polymethylmethacrylate (Polymethylacrylate), polycarbonate, polystyrene, polysulfones (polysulphone), polyhydroxy acid (polyhydroxyacid), poly-acid anhydrides, poe (polyorthoester), polyphosphonitrile, poly phosphate (polyphosphate), polyester, nylon or its mixture etc.Usually the culture vessel that makes with this material will carry out surface treatment in order to the hydrophilic base surface to be provided after cast molding, thereby improves the possibility of effective cell attachment.Surface treatment can take top coat to handle maybe can relate to the mode of wishing to produce at polymer surfaces chemical group on the surface by the use of directional energy.These chemical groups have general affinity to water, perhaps show enough polarity and make its stable other polar group that is adsorbed to.These functional groups cause wetting ability and or increase surperficial oxygen and think and can improve the character of cell in the growth of the substrate surface of this modification.This chemical group can comprise as amine, acid amides, carbonyl, carboxylic acid, ester, hydroxyl, sulfydryl etc.The example of directional energy comprise atmospheric electricity corona (atmospheric corona discharge), radio frequency (RF) vacuum plasma treatment, direct current glow discharge or Cement Composite Treated by Plasma (as, US 6,617,152).The standing procedure of cultivating attached cell at present comprises the chemical mediator that uses definite ingredients, and is added with ox, the mankind or other animal serums.The serum that adds can also promote cell adhesion by the frosting that the base coating (coating) that is easier to cell adhesion with one deck is handled except nutrient and/or somatomedin are provided.
A kind of substrate surface of compatible cell adhesion as an alternative can be a glass, and randomly, introducing as functional group listed earlier strengthen its hydrophilic surface treated.
Other adhere to substrate surfaces and can handle through top coat and produce, as the coating of the macromolecule surface of above-mentioned polymer or processing.In a limiting examples, coating can relate to suitable polycation, as, for example, poly ornithine or polylysine.
In another example, preferred coating and corresponding substrate, comprise one or more extracellular matrix compositions, as, the ECM protein fibre, ln, collagen protein (preferred I type albumen), glycosaminoglycan (as heparin or Suleparoid), fiber adhesion albumen (fibronectin), gelatin, vitronectin (Vitronectin), elastin, Gu raw albumen (Tenascin), but aggrecanase (aggrecan), agrin (agrin), bone silaoprotein (BoneSialoprotein), cartilage matrix protein (cartilage matrix protein), Fibrinogen, fibulin, Saliva Orthana, nidogen (entactin), osteopontin (osteopontin), proplasmin, restrictin, serglycan (serglycin), SPARC/ osteonectin (osteonectin), versican (versican), thrombospondin 1 (thrombospondin 1), or comprise cadherins, connect albumen (connexin), select the adhesion molecule of plain or its multiple combination.
Preferred embodiment can comprise scleroproein, ln or collagen protein.Other preferred embodiments can relate to the component that comprises the ECM composition, as
Figure A20068004882200281
Basement membrane matrix (BDBiosciences), it is the solubility basilar membrane prepared product that extracts from the EHS murine sarcoma, described EHS murine sarcoma is rich in ECM protein, and wherein main component is a ln, secondly is IV Collagen Type VI, heparan sulfate proteoglycan and nidogen.
Particularly embodiment preferred comprises coating of being made up of collagen protein (especially type i collagen albumen) or the coating that comprises collagen protein (especially type i collagen albumen).
As known to the skilled person, for the ease of subsequently with the density inoculating cell of an expectation, counting cells possibly.Wherein, in the present invention, inoculation back cell can mainly adhere to (as cultivating vessel) on the substrate surface that exists in the culture systems, with every mm 2Or cm 2The cell number of described substrate surface inoculation is represented inoculum density.Among the present invention, the inoculum density of the primary cell that obtains from isolating liver or its part is at 1 cell/mm 2With 1 * 10 6Individual cell/mm 2Between, as 1 * 10 1With 1 * 10 5Individual cell/mm 2Between or 1 * 10 2With 1 * 10 5Individual cell/mm 2Between, as 1 * 10 3With 1 * 10 5Individual cell/mm 2Between, 5 * 10 3With 5 * 10 4Individual cell/mm 2Between, 1 * 10 1With 1 * 10 3Individual cell/mm 2Between, 1 * 10 2With 1 * 10 4Individual cell/mm 2Between, as about 1 * 10 1, 5 * 10 1, 1 * 10 2, 5 * 10 2, 1 * 10 3, 5 * 10 3, 6 * 10 3, 7 * 10 3, 8 * 10 3, 9 * 10 3, 1 * 10 4, 2 * 10 4, 3 * 10 4, 4 * 10 4, 5 * 10 4Or 1 * 10 5Individual cell/mm 2
In typical case, after the former generation liver cell inoculation, make cell suspension contact adhesive surface so that allow cell to adhere to described substrate from cell mass.Former generation liver cell with adhere to the contacting of substrate, can be conveniently with cell suspension in the environment that contains matrix at least, in the method for the present invention, be fluid matrix in typical case, it helps the survival and/or the growth of cell.Can with matrix before introducing cell, simultaneously or add system afterwards.Matrix can be fresh, that is: before be not used for culturing cell maybe can comprise at least a portion by cell cultures in advance (as, cultivate to be about to bed board or existing before cell, or to cultivate the cell that cell with inoculation is not too relevant or have nothing to do) part of institute's adaptive processing (conditioned).
For the ease of described adhesion, in one embodiment, the primary cell suspension can contact with adhesive surface at least about 0.5 hour, as, at least about 1 hour, preferably at least about 2 hours, as, at least about 4 hours, more preferably at least about 8 hours, as, at least about 12 hours, even more preferably at least about 16 hours, as, at least about 20 hours, most preferably at least about 24 hours or longer time, as, at least about 28,32,36,40,44 or 48 hours.
In other preferred embodiments, the primary cell suspension can contact between about 2 hours and about 48 hours with adhesive surface, according to appointment between 12 hours and about 48 hours, between preferred about 12 hours and about 36 hours, according to appointment between 16 hours and about 32 hours, even more preferably from about between 20 hours and about 28 hours, most preferably from about 24 hours.
Although the preferred above-mentioned time, also can provide the adhesion that the shorter or longer time is used to be suitable for cell of the present invention, and those skilled in the art can optimize this time.
Make from former generation liver cell group's cell adhesion to above-mentioned adhesion substrate, NA material is removed from culture systems.NA material include, but not limited to not adhere to adhere to suprabasil cell (for example, as, be not inclined to adherent cell, or in the time that allows adherent cell not), do not have cell vigor or dying, cell debris etc.Can remove NA material by from system, discarding matrix in typical case.Adherent cell still sticks in the substrate, randomly uses suitable matrix or isotonic buffer solution (as PBS) rinsing once or repeat rinsing attached cell and culture systems.At this, select to adhere to substrate surface, be used for further cultivation from former generation liver cell group's cell.
Bed board cell and allow the environment of cell adhesion can comprise at least a matrix, fluid matrix normally in the method for the present invention, its sustenticular cell survival and/or growth.Can with matrix before introducing cell, simultaneously or add system afterwards.Matrix can be fresh, that is: before be not used for culturing cell maybe can comprise at least a portion by cell cultures in advance (as, cultivate to be about to bed board or existing before cell, or to cultivate the cell that cell with inoculation is not too relevant or have nothing to do) part of institute's adaptive processing (conditioned).
Matrix can be the suitable culture medium of describing in this specification sheets.The composition of preferred substrate can have identical characteristics, can form identical or basic identical with used matrix in the step of cultivating attached cell subsequently.Perhaps, matrix can be different.Advantageously, matrix can comprise serum or blood plasma, and this can further promote cell adhesion.
Cultivation is from the primary cell of liver organization
Adhere to the cell from the primary cell group of described substrate, preferably in described environment, continue to cultivate at least 7 days, as, at least 8 days or at least 9 days, preferably at least 10 days, as, at least 11 days or at least 12 days, at least 13 days or at least 14 days, more preferably at least 15 days, as, at least 16 days or at least 17 days, or even at least 18 days, as, at least 19 days or at least 20 days or more than.Term " cultivation " is well known in the art, and relates generally to keeping and/or growing of cell and/or its offspring.
In some embodiments, can cultivation of primary cells between about at least 10 days and about 40 days, preferably at least about between 15 days and about 35 days, as, between 15 days and 20 days, as at least about 15,16,17,18,19 or 20 days.Preferably, so cultivation of primary cells is no more than 60 days, or is no more than 50 days, or is no more than 45 days.
Those skilled in the art will know that the cell cultures that prolongs can need regularly substratum to be replaced by fresh culture in culture systems.Those skilled in the art can judge whether to need to change substratum by checking cell cultures parameter (as the outward appearance of pH value, cell density or cell).In typical case, can regularly replace matrix usually, as, every 1 to 10 day, preferred postvaccinal 16 to 32 hours (as, about 24 hours) between, preferably every 2 to 6 days, or more preferably every 2 to 4 days, as, approximately every 2,3 or 4 days.Can change the matrix of whole volumes or removable parts matrix only randomly, thereby keep that part of matrix of being adjusted by previous cell cultures.In one embodiment, the matrix of all volumes all is replaced by fresh matrix substantially.In a further preferred embodiment, during the cell cultures that prolongs, do not change matrix.
There are cultivation of primary cells suspension and further adherent cell under the liquid nutrient medium condition.In typical case, matrix comprises basic medium prescription well known in the art.Many basic medium prescriptions (can be from as American Type Culture Collection, ATCC; Or Invitrogen company, the Carlsbad, the California obtains) can be used for cultivating primary cell herein, include but not limited to Eagle ' s Minimum Essential Medium (MEM), Dulbecco ' sModified Eagle ' s Medium (DMEM), alpha modified Minimum EssentialMedium (alpha-MEM), Basal Medium Essential (BME), Iscove ' s ModifiedDulbecco ' s Medium (IMDM), the BGJb substratum, F-12N utrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle ' s Medium (EMEM), RPMI-1640, Medium 199, Waymouth ' s MB 752/1 or WilliamsMedium E and improvement or combination.The composition of above-mentioned basic medium is being known in the art, and is necessary to improve or adjusts matrix and/or fill-in concentration at cultured cells, also within those skilled in the art's limit of power.In one embodiment, preferred basic medium prescription can be Williams Medium E, and it is a kind of nutritious prescription, is reported in externally can keep adult liver cell culture.Other embodiments can adopt other basic medium prescriptions, as are selected from above-mentioned.
This basic medium prescription comprises the neccessary composition that mammalian cell is grown, and this itself is well-known.For example but unrestricted, these compositions can comprise that inorganic salt (particularly contain sodium, potassium, magnesium, calcium, chlorine, phosphorus and can be the salt of copper, iron, selenium and zinc), physiological buffer (as HEPES, supercarbonate), Nucleotide, nucleosides and/or nucleic acid base, ribose, ribodesose, amino acid, VITAMIN, antioxidant (as gsh) and carbon source are (as glucose, pyruvic acid, for example, Sodium.alpha.-ketopropionate, acetic acid are as sodium-acetate) etc.Obviously, many available matrix are the low-glucose prescriptions that contain or do not contain Sodium.alpha.-ketopropionate.
Be used for cultivating, basic medium can be added with a kind of and multiple other compositions.For example, extra replenishing can provide necessary trace element and the material that is used for the suitableeest growth and amplification to cell.This replenishing comprises Regular Insulin, Transferrins,iron complexes, selenium salt and combination thereof.These compositions can be included in the salts solution, as, but be not limited to Hanks ' balanced salt solution (HBSS), Earle ' s salts solution.Can add other antioxidant supplements, as beta-mercaptoethanol.Because many base matrix have contained amino acid, some amino acid can replenish afterwards, and as L-glutaminate, it is so unstable in solution as everyone knows.Matrix can further be supplemented with microbiotic and/or antifungal compound, as typically, the mixture of penicillin and Streptomycin sulphate and/or other compounds, such as but not limited to, amphotericin, penbritin, gentamicin, bleomycin, Totomycin, that mycin, mitomycin, mycophenolic acid, Nalidixic Acid, Xin Meisu, nystatin, paromycin, polymyxin, tetracycline, Rifampin, spectinomycin, tsiklomitsin, tylosin and zeocin.
Hormone can also be advantageously used in cell cultures, includes but not limited to D-aldosterone, stilboestrol (DES), dexamethasone, estradiol, hydrocortisone, Regular Insulin, prolactin, progestogen, Somatostatin/human growth hormone (HGH), thyrotropin, thyroxine, L-thyronine (thyronine), Urogastron (EGF) and pHGF (HGF).Liver cell also can be benefited from the cultivation that has utilized triiodothyronine (triiodothyronine/triiodithyronine), alpha-tocopherol acetic ester and hyperglycemic-glycogenolytic factor.
Lipid and lipid carrier also can be used for replenishing cell culture medium.This lipid and carrier can include, but are not limited to that linolic acid-oleic acid-arachidonic acid, albumin that cyclodextrin, cholesterol, the linolic acid that albumin is puted together, the linoleic plus oleic acid that albumin is puted together, unconjugated linolic acid, albumin put together put together with unconjugated oleic acid or the like.Albumin can be used for the prescription of FAF similarly.
It is also conceivable that and in cell culture medium, replenish mammiferous blood plasma or serum.Blood plasma or serum often contain vigor and necessary cytokine of amplification and composition.It is suitable to serum also can to consider to use.
Term " blood plasma " is defined as routine.Blood plasma normally obtains from whole blood sample, when extracting blood sample or in the near future, provides or contact anti-coagulant, as heparin, citric acid (as, Trisodium Citrate or acid acid citrate dextrose), oxalic acid or EDTA be in case hemostasis-coagulation.Subsequently, by suitable technology, by centrifugal, the cellular constituent of blood sample is separated from liquid component (blood plasma) in typical case.Therefore term " blood plasma " is meant the composition that does not constitute the mankind or an animal body part.
Term " serum " is defined as routine.Serum normally obtains from whole blood sample, at first allows sample generation blood coagulation, subsequently by suitable technology, passes through centrifugally in typical case, and the cellular constituent of formed grumeleuse and blood sample is separated from liquid component (serum).Inert catalyst as granulated glass sphere or glass powder, can promote blood coagulation.Advantageously, can pass through serum separator (serum-separating vessel well known in the art; SST) preparation serum, described SST contains the inert catalyzer and promotes blood coagulation, also comprises the glue of density setting between the density of centrifugal back liquid component and grumeleuse and cellular constituent, thereby has simplified separation.As an alternative, can obtain serum by removal antithrombotics and scleroproein from blood plasma.Therefore term " serum " is meant the composition that does not constitute the mankind or an animal body part.
Isolating blood plasma or serum can be directly used in method of the present invention.In the method for the present invention, also can properly preserve isolating blood plasma or serum and give over to back usefulness.Can preserve blood plasma or serum in the short period of time in typical case, as, reach about 1-2 week, be higher than blood plasma or serum separately zero pour but be lower than under the room temperature of surrounding enviroment temperature preserve.Usually, about 15 ℃ or following of this temperature, preferred about 10 ℃ or following, more preferably from about 5 ℃ or following, as, about 5 ℃, 4 ℃, 3 ℃, 2 ℃ or about 1 ℃, most preferably from about 5 ℃ or about 4 ℃.As an alternative, blood plasma or serum can be stored in and be lower than the temperature of zero pour separately, promptly freezing preservation.Common as this area, the favourable temperature that is used for freezing preservation blood plasma or serum can be approximately-70 ℃ or following, as, about-75 ℃ following or about-80 ℃ or following.This temperature can favourablely prevent that the blood plasma or any of serum that preserve from thawing, thereby keeps its quality.No matter how long blood plasma or serum need preserve period, can adopt freezing preservation.But when longer-term stored if desired, as surpassing a couple of days or surpassing 1-2 week, freezing preservation was then especially suitable.
Before preservation or the use, can isolating blood plasma of heat inactivation or serum.Heat inactivation is mainly used in the removal fill-in in the art.Hot deactivation generally includes, allow blood plasma or serum be cooled to envrionment temperature gradually after, blood plasma or serum were hatched under 56 ℃ 30 to 60 minutes, as 30 minutes, and lasting mixing.Those skilled in the art will know that any conventional the improvement and demand of said process.
Randomly, also can before preservation or use, blood plasma or serum be sterilized.Common sterilization means can comprise, as, filter by the strainer of one or more apertures less than 1 μ m, preferably less than 0.5 μ m, as less than 0.45 μ m, 0.40 μ m, 0.35 μ m, 0.30 μ m or 0.25 μ m, more preferably 0.2 μ m or littler, as 0.15 μ m or littler, 0.10 μ m or littler.
Be used for suitable serum of matrix of the present invention or blood plasma and can comprise human serum or blood plasma; serum or the blood plasma of non--mankind's animal; preferably non--human Mammals; for example; as, serum or the blood plasma of inhuman primate (as mongoose lemur, monkey, orangutan), tire ox or adult ox, horse, pig, sheep, goat, dog, rabbit, mouse or rat etc.In another embodiment, the present invention has predicted the purposes of the arbitrary combination of above-mentioned blood plasma and/or serum.
Therefore, in one embodiment, can from the species organism identical, obtain serum or blood plasma with acquisition liver cell of former generation.In a limiting examples, human serum or blood plasma can be used for cultivating former generation human liver cell.
In a further preferred embodiment, matrix comprises serum or blood plasma, preferred tire ox (calf) serum or blood plasma, more preferably tire ox (calf) serum (FCS or FBS).
In another embodiment, the matrix that is used to cultivate former generation human liver cell comprises bovine serum or blood plasma, preferred tire ox (calf) serum or blood plasma, more preferably tire ox (calf) serum (FCS or FBS).
In one embodiment, matrix comprise about 0.5% and about 40% (V/V) between serum or blood plasma or for serum, preferred about 5% and about 20% (V/V) between, as, about 5% and about 15% (V/V) between, more preferably from about 8% and about 12% (V/V) between, as, about 10% serum or blood plasma or for serum, the serum of especially preferred above-mentioned definition or blood plasma.
In other preferred embodiments, the matrix that is used to cultivate former generation human liver cell comprises, amount be about 0.5% and about 40% (V/V) between, preferred about 5% and about 20% (V/V) between, as, about 5% and about 15% (V/V) between, more preferably from about 8% and about 12% (V/V) between, as, about 10% bovine serum or blood plasma, preferred tire ox (calf) serum or blood plasma, more preferably tire ox (calf) serum (FCS or FBS).
In other embodiments, matrix can comprise blood plasma or the serum from more than one species.For example, matrix can comprise from the corresponding species of former generation liver cell, the serum of another species or the mixture of blood plasma cultivated.For example, the matrix that is used for cultivator liver cell of former generation can comprise the mixture of human plasma or serum, preferred human serum, and Ox blood plasma or serum, preferred bovine serum.
In addition, matrix can preferably comprise the somatomedin that at least a exogenous (promptly except blood plasma or serum) adds.The technician knows the basic medium conventional ingredient of (adding before serum or the blood plasma), as, especially physiological saline, damping fluid, inorganic salt, amino acid, carbon source, VITAMIN, antioxidant, pH indicator and microbiotic are not considered to somatomedin or differentiation factor in the art.On the other hand, serum or blood plasma are a kind of compositions of complexity, can comprise one or more such somatomedin or differentiation factors.
Term used herein " somatomedin " is meant what a class influenced various kinds of cell type propagation, growth, differentiation, survives and/or moves, can influence the biologically active substance of organism growth, form and changes of function, and it acts on separately or regulated by other materials.Somatomedin usually as part by in conjunction with the acceptor of the response somatomedin that exists in the cell (as, surface or intracellular receptor) and play a role.Somatomedin herein can especially comprise the protein entity of one or more polypeptide chain.
For example, rather than restriction, term " somatomedin " comprises fibroblast growth factor (FGF) family, the family of Delicious peptide (BMP), platelet derived growth factor (PDGF) family, transforming growth factor-beta (TGF-β) family, nerve growth factor (NGF) family, Urogastron (EGF) family, somatomedin (IGF) family that Regular Insulin is relevant, pHGF (HGF) family, hemopoieticgrowth factor (HeGF), Platelet-derived Endothelial Cell Growth Factor (PD-ECGF), angiogenin, vascular endothelial growth factor (VEGF) family, the member of glucocorticosteroid etc.
In the preferred embodiment, matrix contains a kind of somatomedin, and it is Urogastron (EGF) family member.In another embodiment, described EGF family member is selected from: amphiregulin (amphiregulin), β cytokine, EGF, epiregulin, HB-EGF (heparin is in conjunction with epidermal growth factor-like growth factor), NRG1 (neuroregulation element-1) obform body (isoform) GGF2, NRG1 obform body SMDF, NRG1-α, NRG1-β, TGF α, tomoregulin-1 and TMEFF2.At one particularly in the embodiment preferred, matrix comprises EGF.
In another embodiment, matrix comprises a kind of somatomedin, and it is relevant somatomedin (IGF) family member of Regular Insulin.In another embodiment, described IGF family member is selected from: Regular Insulin, IGF1A (type-1 insulin like growth factor A), IGF1B, IGF2, INSL3 (Insulin-Like 3), INSL5, INSL6 and Relaxin.At one particularly in the embodiment preferred, matrix comprises Regular Insulin.
In another embodiment, matrix comprises a kind of somatomedin, and it is a glucocorticosteroid.In another embodiment, described glucocorticosteroid is selected from: dexamethasone, hydrocortisone, Ultracortene-H, prednisone, Methyllprednisolone (methylprednisolone), prednisone, Triamcinolone Acetonide, Kendall compound, fluocinolone acetonide, cortisone, Betamethasone Valerate.At one particularly in the embodiment preferred, matrix comprises dexamethasone.
In another preferred embodiment, matrix comprises the combination of the somatomedin of any two or more exogenous interpolations or the defined preferred growth factor herein.For example, rather than restriction, matrix can comprise EGF and Regular Insulin, or EGF and dexamethasone, or Regular Insulin and dexamethasone, or independent EGF, Regular Insulin and dexamethasone; The matrix that comprises the somatomedin of these exogenous interpolations can preferably include serum or the blood plasma that defines in the above-mentioned embodiment.
The particular growth factor can be induced especially the effect to the cell of vitro culture under what concentration, and this concentration can be used for above-mentioned somatomedin, and this to those skilled in the art concentration is general knowledge.For example, rather than restriction, in typical case can be approximately between 0.1ng/ml and the 1 μ g/ml, between preferred 1ng/ml and the 100ng/ml, as, use EGF under about 25ng/ml concentration; In typical case can be between about 0.1 μ g/ml and 1mg/ml, between preferred about 1 μ g/ml and the 100 μ g/ml, as, use Regular Insulin under about 10 μ g/ml concentration; Can preferably, use dexamethasone under the 10nM concentration according to appointment at about 0.1nM and 1 μ M in typical case at about 1nM and 100nM.
In the preferred embodiment, especially in the method that is used for the human liver cell, the used somatomedin of the present invention can be a human growth factor.Term " human growth factor " is meant the essentially identical somatomedin of a kind of and naturally occurring human growth factor as used herein.For example, when somatomedin was the protein entity, its composing type peptide or polypeptide can have the amino acid primary sequence consistent with naturally occurring human growth factor.The preferred human growth factor who uses the inventive method estimates that this somatomedin causes the effect of the cellular function of expectation.
As described, the inventor has been found that by lasting and cultivates former one period of liver cell generation (period as defined above), the above-mentioned matrix of preferred use is formed, progenitor cell of the present invention or stem cell and propagation occur, and the advantage of the liver cell type of differentiation in the former generation liver cell cultivation that prolongs reduces.Not limited by any hypothesis, former generation liver cell type of differentiation is passable, for example, prolongs the propagation of failing when cultivating, dead and/or contrary differentiation (retro-differentiation).As implement part and describe in detail, can pass through, wherein, other cell types that exist in differentiating forms progenitor cell or stem cell and the primary cell culture, knowledge according to the inventor, described form is expressed as mesenchyme or mesenchyme sample form, in typical case, comprises flat pattern, tangible tenuigenin and/or has the ovogonium of one or two kernel to examine.
The inventor also finds and can further promote described progenitor cell or stem cell former be commissioned to train foster appearance, propagation and enrichment by changing culture medium, thereby help further eliminating the liver cell type of one or more differentiation, especially liver cell, liver cell can occupy leading in isolating former generation liver cell culture.In this case, progenitor cell of the present invention or stem cell can advantageously breed and become the predominant cell types in the former generation liver cell cultivation.As, weakening or be eliminated in the training period because the cell type of one or more differentiation is physiological, the liver cell type of differentiation can be lost; As an alternative, because the contrary in the training period differentiation of one or more cell types, the phenotype of differentiation can be weakened.
When keeping the propagation of progenitor cell of the present invention or stem cell (for example, can easily judge at the visual inspection cell cultures of passing through of different cell types), can use any especially hepatocellular matrix of liver cell type that helps eliminating one or more differentiation.For example, as set forth in the present invention, a kind of variation can be to use the basic medium that contains high glucose concn, as concentration between 3000mg/l and 6000mg/l, between preferred 4000mg/l and the 5000mg/l, and about in typical case 4500mg/l.Another kind of variation can be not have the exogenous interpolation somatomedin of (that is, except existing in serum or the blood plasma).For example, the Regular Insulin, dexamethasone and/or the EGF that do not have exogenous interpolation in the matrix at least.Another kind of variation can be to use the basic medium in addition except that Williams Medium E (especially being fit to long-term cultivation former generation liver cell type, especially liver cell).For example, rather than restriction, the use as the basic medium of MEM, DMEM, alpha-MEM or EMEM can have superiority.Need be appreciated that, can change matrix by above-mentioned a kind of, more than one or all modes.Need also to be appreciated that matrix comprises above-mentioned serum or blood plasma or for serum, comprising the preferred embodiment that describes in detail above.
In one embodiment, can add the matrix that helps eliminating the liver cell type of above-mentioned one or more differentiation and promote progenitor cell of the present invention or stem cell at the beginning at cultivation liver cell of former generation.In another embodiment, can during prolonging cultivation liver cell of former generation, change matrix in this way.For example, can be after inoculation former generation liver cell about 1 day, about 2 days, 3 days, as, about 4 or 5 days, or originate in about 6 days, for example, as, about 7 or 8 days, or originate in about 9 days, for example, as, about 10 or 11 days, originate in about 12 days, as, about 13 or 14 days, more preferably originate in about 15 days, as, about 16,17,18,19 or 20 days according to this mode change matrix.In an exemplary, can be between postvaccinal 16 to 32 hours, 24 hours according to appointment, mode changed matrix according to this.In one embodiment, after mode changes matrix according to this, can as, every 2 to 6 days, preferably every 2 to 4 days, as, every 3 or 4 days replacing/renewal matrix.
Therefore, in an exemplary preferred embodiment, can in the matrix that helps former generation liver cell survival, cultivate former generation liver cell after the bed board, in described former generation,, liver cell comprised the liver cell type of differentiation, as liver cell, when above-mentioned time, matrix can be changed into and help to eliminate the matrix that one or more comprise hepatocellular differentiation liver cell type and promote progenitor cell of the present invention or stem cell.For example, liver cell can be incubated at earlier and have in a kind of, the matrix more than a kind of or all following features former generation: comprise a kind of nutritious basic medium, for example, as WilliamsMedium E, comprise low concentration glucose, as between 500mg/l and 2999mg/l, in the middle of preferred 1000mg/l and the 2000mg/l, the somatomedin that comprises at least a exogenous interpolation, among preferred Regular Insulin, dexamethasone and the EGF a kind of, more than a kind of or all.After this, in above-mentioned period, matrix can be changed into comprise a kind of, more than a kind of matrix of or all following features: comprise the basic medium except that Williams Medium E, for example, basic medium as MEM, DMEM, alpha-MEM or EMEM, comprise height (concentration) glucose, do not contain the somatomedin of exogenous interpolation or do not contain dexamethasone, Regular Insulin and/or EGF at least.Need be appreciated that above-mentioned matrix will comprise above-mentioned serum or blood plasma or for serum, comprising the preferred embodiment that describes in detail above.
As described, above-mentioned former generation liver cell cultivation cause generation and the propagation of in culture progenitor cell of the present invention or stem cell.Can keep described cultivation easily fully breeds until progenitor cell of the present invention that occurs or stem cell.For example, can continue described cultivation and arrive certain degree of converging until cell mass, as at least 40%, preferably at least 50%, more preferably at least 60% and even more preferably at least 70%, as, at least 80%, or at least 90% or above converging.Term used herein " converges " density that is meant culturing cell, and the cell that contacts with each other under this density covers all substantially can be for the surface (that is, converging fully) of growth.
Passage
After the generation and propagation of above-mentioned former generation liver cell cultivation and progenitor cell of the present invention or stem cell, so the cell mass that obtains can go down to posterity once at least.
The inventor finds pleasantly surprisedly, appears at progenitor cell of the present invention or stem cell basic its multiplication capacity that keeps after going down to posterity in the primary cell culture, thereby allows the cell mass of further these cells of enrichment easily.For the purpose of convenient, this stage of described method carries out goes down to posterity and is meant " going down to posterity first " (going down to posterity 1) in the inventive method herein.Cell can go down to posterity at least once, preferably twice or more than twice.Herein, at every turn the going down to posterity after 1 of going down to posterity increases by one with number to be represented, as goes down to posterity 2,3,4,5 or the like.
When going down to posterity, culturing cell dissociates each other (detach) and separates from culture medium.Can carry out dissociating of cell and separate according to (method) well known in the art, (as be selected from trypsinase, collagenase as carry out the enzyme processing with proteolytic enzyme, as I, II, III or IV type, Dispase, pronase (pronase), papoid etc.), with the divalent ion sequestrant (as, EDTA or EGTA) or the mechanicalness processing (as, by the small-bore pipettor or move the liquid head and blow and beat repeatedly), or the arbitrary combination of these processing is handled.Preferably, culturing cell dissociates and separates the individual cells that will produce significant proportion.For example, 40% or the cell of above recovery be individual cells, as at least 50%, preferably at least 60%, as, at least 70%, more preferably at least 80%, as, at least 90% or to have the cell of 95% recovery at least be individual cells.In addition, remaining cell can with cell mass or bunch form exist, wherein major part can contain less relatively cell, as, on average more than the cell between 1 to 10, as be less than 8 cells, preferably be less than 6 cells,, as be less than 3 or be less than 2 cells more preferably less than 4 cells.
In general, a kind ofly be suitable for dissociating and the method for cell dispersion should keep cell viability.Preferably, dissociate and the cell suspension that disperses the back to obtain can comprise at least 60% viable cell, as, at least 70%, more preferably at least 80%, at least 90% to 100% viable cell most preferably.Those skilled in the art will know which kind of condition of common selection guarantees the cell dissociation and the dispersion of expected degree, preserve cell viability.
Afterwards, will dissociate and isolated cells (in typical case, the suspension in isotonic buffer or the matrix) renewed vaccination makes cell adhesion thereon to matrix, and be incubated at subsequently as mentioned above to keep progenitor cell of the present invention in the matrix or stem cell further breeds.In typical case, with 1 * 10 1With 1 * 10 6Individual cell/cm 2Between, as 1 * 10 2With 1 * 10 6Individual cell/cm 2Between, preferred 1 * 10 3With 1 * 10 5Individual cell/cm 2Between, as, about 1 * 10 3Individual cell/cm 2, about 5 * 10 3Individual cell/cm 2, about 1 * 10 4Individual cell/cm 2, about 5 * 10 4Individual cell/cm 2, or about 1 * 10 5Individual cell/cm 2, preferred about 1 * 10 3With 1 * 10 4Individual cell/mm 2Between inoculum density renewed vaccination cell.
As an alternative, between about 1/8 and 1/2, between preferred about 1/4 and 1/2,1/2 or about 1/3 ratio of division (splitting ratio) renewed vaccination cell more preferably from about.Ratio of division is represented the ratio of the cell that goes down to posterity, is seeded to it in empty (in typical case, new) culture vessel and the identical surf zone of container of acquisition cell.
Again the adhering substrate of bed board cell is as describing in detail in this explanation.Matrix can be preferably with the matrix phase of bed board liver cell of former generation with (preferred embodiment that comprises above-mentioned this matrix) or different.Preferred this matrix is collagen protein, especially above-mentioned type i collagen albumen.
Further cultivating the cell that goes down to posterity becomes at least 50% until cell and converges, as, at least 60%, preferably at least 70%, example, at least 80%, more preferably at least 90%, as, have 95% or even converge fully at least.
The inventor has been found that cell mass that this of described method obtains in stage comprises the progenitor cell of the present invention and the stem cell of significant proportion, and cell can advantageously go down to posterity once (that is: second pass generation) at least at least again, and go down to posterity the aforesaid substantially first time.Judge that by form and/or molecular marker this has further increased progenitor cell of the present invention and the ratio of stem cell in cell mass, even obtains the progenitor cell of the present invention of basic homogeneous and the group of stem cell.
Therefore, according to the present invention, inoculation and cultivating after former generation liver cell, cause progenitor cell or generation and the propagation of stem cell in described culture, cultured cells goes down to posterity at least once (promptly, go down to posterity first) preferably at least twice (promptly for the first time and second pass generation), randomly more times go down to posterity (i.e. the first time, the second time and follow-up go down to posterity) at every turn.For example, inoculate after former generation liver cell, cell can go down to posterity at least once, at least 2 times, at least 3 times, at least 4 times or at least 5 times.In another embodiment, inoculate and cultivate after former generation liver cell, cell can go down to posterity between 2 to 10 times, as, between 2 to 8 times, or between 2 to 5 times.Going down to posterity with the first time, as above-mentioned, basically identical or similarly carry out under the condition extra going down to posterity (as, cell dissociation and dispersion, renewed vaccination, matrix etc.) and cultivate (as, matrix, matrix changes, final converge etc.), comprising its preferred embodiment, and comprise conspicuous to those skilled in the art improvement.
Therefore method of the present invention provides the cell mass as defined progenitor cell of the disclosure or stem cell that comprises significant proportion, and the one or many that the ratio of described progenitor cell or stem cell is cultivated along with former generation liver cell prolongation goes down to posterity and increases.In typical case, described group will comprise at least about 10%, as, at least about 20% described progenitor cell or stem cell, but the contriver finds will obtain usually more a high proportion of described progenitor cell or stem cell, as, at least 30%, at least 40%, at least 50%, be at least 60%, at least 70%, at least 80% at least at least 90% or more than.In addition, method even can produce the described progenitor cell or the population of stem cells of basic homogeneous or homogeneous.Can estimate the ratio of progenitor cell or stem cell by any suitable standard method, as pass through flow cytometer.
Keep the cell of acquisition
When obtain to contain the progenitor cell of the present invention in liver source or the group of stem cell by method of the present invention, and can so when described progenitor cell of enrichment or stem cell, keep and/or proliferating-cell population under the condition that can next not break up allowing described progenitor cell or stem cell growth and multiplication.This condition can be, as, be used to obtain the condition of progenitor cell or stem cell.Can pass judgment on the those skilled in the art whether cytodifferentiation takes place and to set up other conditions easily.This helps to increase because the available progenitor cell of subsequent use or the number of stem cell.
The inventor have been found that can frozen (as this is known for mammalian cell) former generation or any other follow-up going down to posterity be used for subsequently purposes.
The inventor has been found that progenitor cell of the present invention or the stem cell basic multiplication capacity that keeps after freeze thawing that occurs in the primary cell culture.Described cell can be preserved with the form of freeze concentration cell suspension, as known in the art, under the same terms as described in this manual, be thawed and bed board (cell) again.
Progenitor cell of the present invention or stem cell
The inventor find by cultivate as mentioned above and preferred also by go down to posterity former generation liver cell obtain cell mass comprise the progenitor cell or the stem cell in liver of the present invention source, at least a mesenchyme mark of its coexpression (promptly being positive) to following, especially for example CD90, CD73, CD44, a kind of in vimentin and the α-smooth muscle actin (ASMA), more than a kind of, as 2,3, or 4 kinds or whole, and liver cell mark albumin (ALB) and one or more plant other livers or liver cell mark possibly, preferred CD29, alpha-fetoprotein (AFP), a kind of in α-1 antitrypsin and/or the MRP2 translocator, more than a kind of or whole.
In a more particular embodiment, the progenitor cell in liver of the present invention source or stem cell can at least a mesenchyme marks of coexpression (promptly being positive to following), especially CD90, CD73, CD44, a kind of in vimentin and the α-smooth muscle actin (ASMA), more than a kind of, as 2,3, or 4 kinds or whole, and liver cell mark albumin (ALB) and one or more other liver cell marks possibly, in preferred α-1 antitrypsin and the MRP2 translocator one or both, and at least a liver landmarks thing CD29 or alpha-fetoprotein (AFP).
That any described adult hepatic progenitor cell or stem cell can further be expressed is a kind of, more than a kind of or all following molecules that indicating liver cell-sample character or function: G6P, CYP1B1, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2.That described adult hepatic progenitor cell or stem cell also can have is a kind of, more than a kind of or whole following feature: hematopoiesis mark CD45 and CD34 are negative at least, also can be that for example CD105 and HLA-DR are negative for one or more other hematopoiesis marks; Bile duct cell epithelium mark cytokeratin-19 (CK-19) feminine gender, more possibly epithelium marks are negative; At least undifferentiated stem cell markers CD117 and Oct-4 are negative, and also can be a kind of or negative more than a kind of embryonic stem cell mark; The alpha-fetoprotein of low expression level (AFP).Preferred described adult hepatic progenitor cell or stem cell can have mesenchyme sample form, especially comprise with individual layer, flat pattern, significantly tenuigenin and/or have in the oval nuclear of one or two kernel a kind of, more than a kind of or whole.
Identify that by its CD (common determinant, " common determinant ") title the specific cells surface molecular is as known in the art.Determined in other names in should using or the use of title such as this area.Also can find other explanations of concrete molecule in an embodiment.
Wherein when thinking that a cell is positive to the special sign thing, this expression, when the suitable contrast of contrast when suitably measuring, those skilled in the art will determine the appearance or the sign of the clear signal of this mark, and will be detectable or detect by reverse transcriptase polymerase chain reaction as antibody.Wherein said method allows this mark of quantitative evaluation, positive cell on average can produce the signal that significantly is different from contrast, as, but it is unrestricted, at least be more than 1.5 times of signal that control cells produces, as, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times even higher.
Can use any suitable immunological technique well known in the art to detect the cell-specific mark, as flow cytometer, immunocytochemistry or affine absorption, Western engram analysis, ELISA etc., or the technology by any suitable survey mark thing mRNA amount, as Northern hybridization, sxemiquantitative or quantitative RT-PCR etc.
Those skilled in the art will know that and to gather in the crops the cell mass that (as by suitable dissociation technique) obtains by present method, comprising above-mentioned progenitor cell or stem cell, and the cell of further alternatively those displaying specific characteristics (by means commonly known in the art) of enrichment (so this cell can be separated from described group).For example, rather than restriction, the cell that presents one or more progenitor cells of the present invention or stem cell surface characterization of molecules, as, the mark that one or more are listed above can be discerned by specificity (label) antibody or other identification agents of anti-this molecule, and never show in the cell of this surface molecular and separate, as by fluorescence-activated cell sorting or utilize avidity to be bonded to, as, post, pearl or surface (plate).Any other method of enrichment of cell is included within the invention scope.
Another aspect, the inventor thereby realized progenitor cell or the stem cell (vertebrates of a kind of novel separation from adult hepatic, preferred mammal, more preferably human cell) method, described progenitor cell or cells and characteristic of stem are at least a mesenchyme mark of coexpression (promptly being positive to following), especially CD90, CD29, CD44, a kind of in vimentin and the α-smooth muscle actin (ASMA), more than a kind of, as 2,3, or 4 kinds or all, and liver cell mark albumin (ALB) and one or more plant other liver cell marks possibly.That described adult hepatic progenitor cell or stem cell also can have is a kind of, more than a kind of or whole following feature: negative to hematopoiesis mark CD45, CD34 and CD117 at least, and can also be negative to one or more other hematopoiesis marks; (CK-19) is negative for cytokeratin-19; Mesenchyme sample form especially comprises with monolayer growth, flat pattern, tangible tenuigenin and/or have a kind of or whole in the oval nuclear of one or two kernel.
Therefore, in a specific embodiments (1), progenitor cell or stem cell coexpression ALB and CD90, CD73, CD44, vimentin and the α-smooth muscle actin (ASMA) in isolating liver source.In another embodiment (2), progenitor cell or stem cell coexpression ALB and α-1 antitrypsin and CD90, CD44, vimentin and the α-smooth muscle actin (ASMA) in liver source.In another embodiment (3), progenitor cell or stem cell coexpression ALB and MRP2 and CD90, CD73, CD44, vimentin and the α-smooth muscle actin (ASMA) in liver source.In another embodiment (4), progenitor cell or stem cell coexpression ALB, α-1 antitrypsin and MRP2 and CD90, CD44, vimentin and the α-smooth muscle actin (ASMA) in liver source.In another embodiment (5), the progenitor cell or the stem cell in any one liver source are also expressed CD29 in the above-mentioned embodiment (1) to (4).In another embodiment (6), the progenitor cell or the stem cell in any one liver source are also expressed alpha-fetoprotein in the above-mentioned embodiment (1) to (4).In another embodiment (7), the progenitor cell or the stem cell in any one liver source are also expressed CD29 and alpha-fetoprotein in the above-mentioned embodiment (1) to (4).
In other embodiments (8), the progenitor cell or the stem cell in any one liver source are CD45 and CD34 feminine gender in the above-mentioned embodiment (1) to (7).In other embodiments (9), the progenitor cell or the stem cell in any one liver source are CD117 and Oct-4 feminine gender in the above-mentioned embodiment (1) to (7).In other embodiments (10), the progenitor cell or the stem cell in any one liver source are CD45, CD34, CD117 and Oct-4 feminine gender in the above-mentioned embodiment (1) to (7).
In other embodiments (11), the progenitor cell or the stem cell in any one liver source are the CK19 feminine genders in the above-mentioned embodiment (1) to (10).In other embodiments (12), the progenitor cell or the stem cell in any one liver source are the CK7 feminine genders in the above-mentioned embodiment (1) to (10).In other embodiments (13), the progenitor cell or the stem cell in any one liver source are CK19 and CK7 feminine gender in the above-mentioned embodiment (1) to (10).
In other embodiments (14), the progenitor cell in any one liver source or stem cell performance mesenchyme sample form in the above-mentioned embodiment (1) to (13) especially comprise with monolayer growth, flat pattern, tangible tenuigenin and/or have the oval nuclear of one or two kernel.
In another embodiment (15), in the above-mentioned embodiment (1) to (14) progenitor cell in any one liver source or stem cell also express a kind of, more than a kind of or all following molecules that indicating the character or the function of liver cell sample: G6P, CYP1B1, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2.
In another embodiment (16), the progenitor cell in any one liver source or stem cell are also expressed a kind of, more than a kind of or all following molecule: CD49e, CD13, CD54, I class major histocompatibility complex (MHC) (HLA-ABC) in the above-mentioned embodiment (1) to (15).
In another embodiment (17), in the above-mentioned embodiment (1) to (16) progenitor cell in any one liver source or stem cell be a kind of, more than a kind of or all following molecule feminine genders: CD105, HLA-DR, CD133, CD49b, CD49f and CD140.
In another embodiment (18), the progenitor cell or the stem cell low expression level alpha-fetoprotein (AFP) in any one liver source in the above-mentioned embodiment (6) to (17).Preferably, the low-level expression level of measuring in the normal liver cell that corresponds essentially to.Preferably, be lower than the middle expression level of measuring of human liver clone (as HepG2) that tumorigenesis is modified.
The progenitor cell or the stem cell in the isolating liver of the present invention source can preferably be showed cells and characteristic of stem, show limited at least (can be unlimited substantially) self especially in typical case, i.e. propagation and the ability of not breaking up.For example, rather than restriction, progenitor cell of the present invention or stem cell can breed goes down to posterity at least for 4 times, as, go down to posterity, go down to posterity at least 10 times or go down to posterity at least 20 times, go down to posterity at least 50 times at least 6 times or more.
Another aspect, the invention provides or direct acquisition obtainable by method isolating adult hepatic progenitor cell or stem cell, comprise clone and/or the cell mass of described adult hepatic progenitor cell or stem cell, described method comprises: (a) preferred two step collagenase method, from object dissociate adult hepatic or its part, preferred vertebrates, Mammals and more preferably human subjects are so that form the primary cell group from described adult hepatic or its part; (b) the primary cell group is inoculated on the matrix that is coated with type i collagen among the WilliamsMedium E, wherein contains foetal calf serum, preferred 10% (V/V), EGF, preferred 25ng/ml, Regular Insulin, preferred 10 μ g/ml, dexamethasone, preferred 1 μ M; (c) allow cell to adhere on the described matrix at least 24 hours, then matrix is replaced by and contains fresh matrix of forming in (b) from the primary cell group; (d) two weeks of culturing cell (preferred 15 days) on matrix described in (c); (e) matrix is replaced by the DMEM that contains height (concentration) glucose and FCS, preferred 10%, continue culturing cell, thereby progenitor cell of the present invention or stem cell are taken place and propagation; (f) randomly and preferably, make cell obtain about 70% converge and cell goes down to posterity once at least, preferably at least 2 times, wherein cell inoculation is cultivated to matrix described in (b) and in matrix described in (e).
Step (a) can preferably include by allowing the cell be the sieve of the 0.25mm primary cell that dissociates through at least one aperture, preferably is decreased to the sieve of 0.25mm gradually by a series of meshes, as, among the embodiment 1.
The matrix of step (e) can preferably not comprise dexamethasone, Regular Insulin and EGF in one embodiment.The somatomedin that does not comprise any exogenous interpolation in another embodiment.
On the one hand, the invention still further relates to aforesaid method.
On the other hand, the invention provides isolating adult hepatic progenitor cell or stem cell according to the obtainable or direct acquisition of operating process of statement among the embodiment 1, comprise clone and/or the cell mass of described adult hepatic progenitor cell or stem cell.
On the other hand, the inventor is by method of the present invention, especially as putting down in writing among the embodiment 1, adult people hepatic progenitor cell or stem cell cell mass (clone) have been set up, and on February 20th, 2006 described isolated cells system is preserved in Belgian microorganism cooperation preservation center (Belgian Coordinated Collections ofMicroorganisms (BCCM/LMBP)) according to budapest treaty (Budapest Treaty), preserving number is that LMBP 6452CB (is authorized by international preservation mechanism; Identify with reference to providing: ADHLSC) by the preservation people.Therefore, an aspect of of the present present invention relates to and is preserved in isolated cells, clone and the cell mass of the BCCM number of including for LMBP 6452CB (herein for " LMBP 6452CB " clone), subbreed comprises clone's subbreed, also relate to its offspring, broken up the offspring comprising it, especially liver cell or liver cell like cell prepared therefrom also relate to the derivative of its genetic modification.
Those skilled in the art will know that the method according to this invention can have biological property from progenitor cell or stem cell, the clone that comprises described progenitor cell or stem cell and the cell mass that the adult human liver obtains, especially with the consistent or similar propagation of the clone of above-mentioned preservation and differentiation capability, cellular form and/or marker expression, although they can have difference (because the normal heritable variation between the people) in the heredity.Therefore, have progenitor cell or the stem cell or the cell mass of or similar biological property consistent,, be also included within the scope of the invention especially from liver with the cell mass of preservation.
Another aspect the invention provides the progenitor cell that comprises isolating adult hepatic or the cell mass of stem cell, and described cell or stem cell have above-mentioned feature, randomly through further modifying, as genetic modification.In one embodiment, described cell mass can comprise about 5% or more than, as, about 10% or above described progenitor cell or stem cell, about 20% or above, about 30% or above, about 40% or above, about 50% or above, about 60% or above, about 70% or above, about 80% or above or about 90% or above progenitor cell or stem cell, or and the group of described progenitor cell or basic homogeneous of stem cell or homogeneous.
On the other hand, the invention provides progenitor cell of the present invention or stem cell, (randomly through further modifying, as genetic modification) and the clone set up by propagation liver source.This propagation can be from a kind of progenitor cell or stem cell (cloned cell line) or from more than a kind of cell.
On the other hand, the invention provides the obtainable or direct acquisition of method (comprising its preferred embodiment) by the invention described above isolating adult hepatic progenitor cell or stem cell, comprise clone and/or the cell mass of described adult hepatic progenitor cell or stem cell.
In a specific embodiments, therefore the invention provides from the method (Fig. 1) of the progenitor cell mesenchyme-sample stem cell of normal (people) liver isolating active of adult.Stem cell can breed in substratum, and expresses the mark of multiple liver cell type, for example, and albumin (liver cell), vimentin (stellate cell), α-smooth muscle actin (ASMA) (Fig. 2 A).They do not have the phenotype of courage, as cytokeratin 19 immunostainings of feminine gender and (Fig. 2 B) shown in the RT-PCR analysis.When utilizing flow cytometer to detect, ADHLSC shows negative to CD45, CD34 and CD117, represents that it is not polluted by the lymph hematopoietic lineage.By contrast, ADHLSC is to CD90, CD29 and CD44, and the mark of mesenchyme system shows positive.
In one embodiment, described cell can keep its multiplication capacity after trypsinase/EDTA handles.Incubated cell allows these cell-specifics to be divided into liver cell (Fig. 3) in the matrix that limits.According to other specific criteria, they can't change and are divided into osteocyte or adipocyte, as utilize special energy (multipotent) people mesenchyme medullary cell viewed.The ability of its propagation, and their liver specificity makes curative effect and security that liver cell is transplanted increase.In addition, because it is derived from adult, these stem cells can avoid embryonic cell relevant immunity, ethics and carcinogenic problem.
The differentiation of progenitor cell of the present invention or stem cell
Except detecting the cell sign thing, the research cell can provide information in differentiation external and/or in vivo.
The inventor finds that also progenitor cell or the stem cell from the liver that obtain by aforesaid method can have the specificity differentiation capability.Particularly, stem cell can be divided into liver cell or liver cell like cell.In another embodiment, described cell is not divided into mesoderm (mesenchyme) cell type, for example, as, osteocyte, chondrocyte, myocardial cell, phoirocyte, Tenocyte cell (tendonocyte), adipocyte or stroma cell (stromal cell).
Isolating adult hepatic progenitor cell of the present invention or stem cell, the clone that comprises described adult hepatic progenitor cell or stem cell and/or cell mass (especially will be pointed out, even so but be not limited to, LMBP6452CB is) or its offspring, can advantageously be divided into cell, especially liver cell or the liver cell like cell of hepatic cell line through inducing.This differentiation can occur in the body external or stripped.Therefore, the present invention also provides the method that produces liver cell or liver cell like cell from isolating progenitor cell of the present invention or stem cell, and liver cell or liver cell like cell that acquisition is provided.
In a specific embodiments, therefore the progenitor cell in the isolating liver of the present invention source or stem cell are characterised in that the ability that it is divided into liver cell or liver cell like cell and lack towards the ability of mesoblastema type (for example, as osteocyte and adipocyte) differentiation.
One skilled in the art will appreciate that can external, exsomatize or body in observe towards the ability of certain specific cell type differentiation or lack this ability.By example, as known in the art, external or stripped by cellular exposure is come evaluate differentiation in the matrix that contains specificity differentiation.Otherwise, can be in vivo assess the differentiation (as, cell transplanting, injection or that use) of (cell) introduced by destiny subsequently.Those skilled in the art can discern the differentiation towards particular cell types by phenotype principle (including but not limited to the expression of cellular form, protein marker and/or specific metabolic activity or other physiology paths).
Being divided into the cell of hepatocyte lineage can be advantageously take place under the condition that cytokine and somatomedin (can be liver-specific) exist.For example, pHGF (HGF) or spreading factor (scatter factor) are the cytokine of known promotion to the liver cell phenotypic differentiation.Similar, the non-limiting tabulation of other cytokines comprises Urogastron (EGF), basic FGF, Regular Insulin, niacinamide, oncostatin (oncostatin) M, dexamethasone, hdac inhibitor (as Sodium propanecarboxylate), dimethyl sulfoxide (DMSO), vitamin A or matrix components, as heparin sulfate, above-mentioned these also participate in hepatocellular differentiation.The inducing hepatocyte differentiation is being known in the art, and can further be optimized by the technician.
The cell that can identify by means commonly known in the art and separate differentiation subsequently from its undifferentiated counterpart (counterpart).For example, by identifying the cell of being induced differentiation making the noble cells number exceed under the condition of undifferentiated cell the selectivity culturing cell.Similarly, the cell that morphological change that does not have by undifferentiated counterpart and feature are identified differentiation, the complicacy that distributes as size, shape or the cell within a cell device of cell.The method of the cell of the evaluation differentiation of also considering is the expression by the specific protein marker, as cell surface marker.Can pass through, as flow cytometer, ELISA and/or magnetic bead, the detection that realizes these cells with separate.Reversed transcriptive enzyme chain reaction (RT-PCR) technology also can be used to gene expression response changes of differentiation.In addition, the full genome analysis of application microarray technology can be used for identifying the cell of differentiation.
The genetic modification of progenitor cell of the present invention or stem cell
Can further modify described adult hepatic progenitor cell or stem cell, as above-mentioned genetic modification.The present invention also comprises the offspring of adult hepatic progenitor cell or stem cell, comprises breaking up the offspring.
In one embodiment, for the progenitor cell of the present invention that improves acquisition or the replication of stem cell, as known in the art, can be with cell telomereization (telomerised).If with the mode genetic modification of TERT in transit cell record and translation, then cell is described to by " telomereization " cell by the nucleic acid of the coding reverse transcriptase of telomere (TERT) of any species.The offspring of the cell that this term also has been used for originally having been transformed, it has inherited the ability with higher level expressing TERT coding region.The TERT encoding sequence is taken from or is transformed the gene from Mammals TERT in typical case, in the following example shown in human and mouse TERT.Can by with the appropriate carriers genetically modified cell with its telomereization, thereby express the composition (TERT) of telomerase catalytic with higher level.Especially suitable is the human telomerase catalyst component (hTERT) that provides as WO1998/14592.For some application, can use other TERT sequences.Also mentioned the additive method of cell immortalityization, (US 5 as the DNA with coding SV40 large T antigen, 869,243, WO 1997/32972), with Epstein Bar virus infection, introduce proto-oncogene such as myc and/or ras, introducing virus replication gene such as adenovirus E 1 a and and immortalized cell line with expectation phenotype merge pair cell and carry out genetic modification.When cell is used for the treatment of purpose, not too be fit to usually with proto-oncogene or tumour virus (oncovirus) product transfectional cell.
Usually, when considering that described cell or its offspring (offspring who comprises its differentiation) when being used for the treatment of, will be introduced into especially human body of human or animal as this cell, preferred no-go end granulation or other immortalization methods are transformed progenitor cell of the present invention or stem cell.
Among the present invention,, before subsequent applications is as treatment or research, can or transform progenitor cell or stem cell or its offspring who obtains with purpose nucleic acid stability or transient transfection from liver as known in the field basically.The purpose nucleotide sequence can include but not limited to, as, those codings improve cell type useful in the treatment and (as are derived from the cell type of progenitor cell of the present invention or stem cell, especially liver cell or liver cell like cell) the sequence of gene product of growth, differentiation and/or function, or delivery of therapeutic gene to this cell is used or the sequence of implant site.
For example, rather than restriction, can transform the progenitor cell of acquisition or stem cell or its offspring and come composing type or induced express polypeptide, described polypeptide is usually by liver cell especially liver cell expression, but in the patient defective or disappearance.This indicia of defects patient's pathological state.But use the cell production of recoverin matter afterwards of transformation like this, thereby help to treat the patient.For example, progenitor cell or stem cell or its offspring can be contained coding metabolizable protein such as ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, carbamyl phosphate synthetase, the N-acetylglutamat synthase, glutamine synthetase, glycogen synthetase, G-6-Pase, succinodehydrogenase, glucokinase, pyruvate kinase, acetyl CoA carboxylase, fatty acid synthetase, alanine aminotransferase, glutamate dehydrogenase, ferritin, low-density lipoprotein (LDL) acceptor, the P450 enzyme, and/or the allogeneic dna sequence DNA of ethanol dehydrogenase.As an alternative, cell can contain the DNA of coding excretory plasma proteins such as albumin, Transferrins,iron complexes, complement, composition C3, alpha2-macroglobulin, Fibrinogen, the XIII factor, the IX factor, alpha1-antitrypsin etc.
Liver is the center that produces a lot of secretory proteins.It connects with the recycle system on dissecting in such a way, promptly allows range protein efficiently to discharge into blood flow.Therefore, the proteinic particular cell types of systematicness effect is arranged,, described encoding gene is inserted in the liver cell of the present invention if when especially being difficult to gene integration to these cells with respect to common generation coding.For example, various hormone genes or specific antibody gene can be inserted into and secrete its gene product in the liver cell of the present invention to the recycle system.
Use traditional gene transfer method that nucleic acid is introduced cell.Introduce a kind of gene really blanking method be not vital for the present invention.For example, the physical method of DNA being introduced cell comprises microinjection and electroporation.Chemical process, as calcium phosphate altogether-precipitation and DNA is mixed liposome also is the standard method of DNA being introduced mammalian cell.Also can consider virus transfection.Use standard vector to introduce DNA, as those from mouse and the retroviral carrier of fowl (as, see Gluzman etc., Viral Vectors, Cold Spring Harbour Laboratory, Cold SpringHarbour, N.Y., 1988).Standard DNA recombination method well known in the art (see, as, Ausubel etc., Current Protocols in Molecular Biology, John Wiley﹠amp; Sons, New York, 1989) and gene therapy developed with virus vector and successfully be used for clinically (seeing, as, Rosenberg, etc., N.Engl.J.Med, 323:3701990).
The purposes of progenitor cell of the present invention or stem cell
The isolating progenitor cell of the present invention that is derived from liver or stem cell, the clone that comprises described progenitor cell or stem cell and/or cell mass can be used for various objectives and (should know following purposes and use any adult hepatic progenitor cell or the stem cell that can extensively consider above-mentioned definition, especially obtainable by the method for above-mentioned definition or the cell of direct acquisition, the cell that shows above-mentioned qualification feature and LMBP 6452CB clone, people's liver stem cells system (ADHLSC) in preferred adult source; Its offspring; Or the derivative of its genetic modification), it includes but not limited to:
-use according to isolating progenitor cell of the present invention or stem cell or its group, carry out liver cell transplants in order to treat liver metabolism defective, liver degenerative disorders (liver degenerative disease) or fulminant hepatic failure (fulminant liver failure)
-use according to isolating progenitor cell of the present invention or stem cell or its group to prepare biology-artificial liver device,
-prepare human liver disease's animal model by in animal, transplanting according to the present invention isolating progenitor cell or stem cell or its group,
-by isolating progenitor cell or stem cell or its group prepare external toxicology, pharmacology animal model according to the present invention,
-testing new drug on isolating progenitor cell or stem cell or its group, comprise the antiviral that is used for human hepatitis virus according to the present invention.
For example, analyze the uPA of spleen transplantation ADHLSC (LMBP 6452CB system) + /+-SCID and SCID mouse liver show that these cells can transplant and be divided into ripe liver cell (Figure 4 and 5).In addition, detect human albumin in the transplanting mice serum after these transplanted for 10 weeks, and do not detect the alpha-fetoprotein level.
Can be used for the transplanting of congenitalDy olism, the animal model of liver failure or the transplanting of human virus's hepatitis animal model according to progenitor cell of the present invention or stem cell (what will say especially is to include but not limited to LMBP 6452CB system certainly).Therefore cell of the present invention can be used for the treatment of liver related disease, includes but not limited to liver failure, hepatitis, congenital metabolic obstacle.
In an exemplary, as shown in Example 1, the inventor has proved, when by intrasplenic injection such as immunity-defective Mammals (more preferably rodent, especially mouse or rat) when using, people's hepatic progenitor cell of the present invention or stem cell keep its multiplication capacity and are implanted in host's liver.Therefore in one embodiment, the group that to contain progenitor cell of the present invention or stem cell, preferably (what will say especially is from the mankind, yes but be not limited to LMBP6452CB system), introduce (as by injection) and permission and implant in the immune deficiency Mammals of heredity or chemical improvement, thereby obtain animal model.Preferably, this population can comprise at least 2 * 10 6Individual cell of the present invention.The technician will know other modes that cell mass of the present invention is induced described liver transplantation from adult hepatic progenitor cell or stem cell of using.
As describing in detail among the embodiment 1, the cell of transplanting does not have hyper-proliferative, thereby has underestimated its advantage in human or other animals (preferred mammal) Transplanted cells.
The present invention comprises that also use is used for following purpose according to isolating progenitor cell of the present invention or stem cell or its group (what will say especially is to include but not limited to LMBP 6452CB system certainly):
-liver transplantation progenitor cell or stem cell are treated the inborn errors of metabolism based on liver: the non-complete example of this disease comprises pku and other amino acid metabolism diseases (aminoacidopathies), hemophilia and other thrombin defectives, familial hypercholesterolemia and other lipid metabolism disorders, urea cycle disorder, glycogenosis (glycogenosis), galactosemia, fructosemia (fructosemia), tyrosinemia (tyrosinemia), protein and carbohydrate metabolism defective, organic aciduria, mitochondrial disease, peroxysome and lysosome are not normal, protein synthesis is unusual, liver cell translocator defective, glycosylation defect etc.
-transplant and treat acquired liver degenerative disease according to hepatic progenitor cell of the present invention or stem cell,
-use and treat fulminant hepatic failure and acute or chronic liver failure according to hepatic progenitor cell of the present invention or stem cell,
-in bioartificial livers device and liver supplementary unit, use according to hepatic progenitor cell of the present invention or stem cell,
-by the human liver disease animal model of transplanting in small-sized and macrofauna according to hepatic progenitor cell of the present invention or stem cell acquisition,
-preparation human hepatitis virus infection animal model (HBV, HAV, HCV, HEV, HDV...) is studied the life history, propagation, resistance, result of treatment, purposes and any use of the antiviral research according to hepatic progenitor cell of the present invention or stem cell transplantation
-use to prepare external toxicology, pharmacology animal model according to hepatic progenitor cell of the present invention or stem cell,
-on progenitor cell according to the present invention or stem cell, test new drug,
-can be by in progenitor cell according to the present invention or stem cell, inserting in the gene therapy of the virus sequence of amplification in vitro,
The metabolic animal model of-research people's liver cell,
-by using, tolerate heterogenote according to progenitor cell of the present invention or stem cell, and/or
-use and avoid, prevent or treat liver or liver cell heteroplastic transplantation rejection according to progenitor cell of the present invention or stem cell.
In one aspect of the invention, progenitor cell of the present invention or stem cell or its offspring comprise and break up the offspring, and intention is used for the treatment of application, as, organizational project and cell therapy.
Those skilled in the art understand purposes described in detail herein and can comprise progenitor cell or stem cell, comprise the clone of progenitor cell or stem cell and the purposes of cell mass, and its offspring, comprise the offspring of differentiation, especially liver cell or liver cell like cell, or the purposes of its genetic modification derivative.
As mentioned above, be derived from the progenitor cell or the stem cell of liver, the clone that comprises progenitor cell or stem cell and cell mass (what will say especially is according to the present invention, include but not limited to LMBP 6452CB system) or its offspring (randomly genetic modification), can be used for the cell replacement treatment.Can come supplementary functions sexual cell or replacement to lose the cell of function to the destination organization dosed cells of object.As an alternative, also can consider to provide the method for noble cells (especially liver cell or liver cell like cell), wherein in the presence of differentiation factor, make progenitor cell or differentiation of stem cells, separate and be applied to object.
The morbid state or the defective that are characterised in that liver quality (mass) and/or loss function can be benefited from progenitor cell of the present invention or stem cell, comprise listed above those, also include but not limited to alagille syndrome, alcoholic liver disease (alcohol is induced liver cirrhosis), alpha-1-amtitrypsin deficiency (all phenotypes), hyperlipidaemia and other lipid metabolism disorders, autoimmune hepatitis, Bu-Jia syndrome (Budd-Chiari syndrome), Biliary atresia, I, II and carrying out property of III type familial cholestasis, liver cancer, the Caroli disease, crigler-najjar syndrome, fructosemia, galactosemia, glycosylated defective, other carbohydrate metabolism disturbances, carbohydrate deficiency (carbohydratedeficient), sick and other peroxysome diseases of refsum, Niemann Pick disease, sick and other lysosomal diseases of wolman, tyrosinemia, triple H and other disorders of amino acid metabolism, Dubin Johnson syndrome, fatty liver (nonalcoholic fatty liver disease), Gilbert syndrome, glycogen storage disease I and III, hemochromatosis, A-G type hepatitis, porphyria, primary biliary cirrhosis, sclerosing cholangitis, tyrosinemia, the thrombin defective, hemophilia B, pku, Wilson's disease (Wilson ' s Disease), fulminant hepatic failure, liver failure behind the hepatectomy, the mitochondrial respiratory chain disease.In addition, described cell also can be used to treat the hepatopathy that virus infection causes.
Therefore, one aspect of the present invention provides adult hepatic progenitor cell of the present invention or stem cell, the clone that contains described progenitor cell or stem cell or cell mass (will say especially, include but not limited to, LMBP6452CB is) or its offspring (offspring who comprises differentiation, especially liver cell or liver cell like cell are randomly as detailed above through genetic modification) purposes of the purposes that is used for the treatment of and/or the preparation medicine that is used for the treatment of hepatic diseases.This disease can comprise the disease that influences hepatic tissue, and especially considering influences the disease of hepatocyte activity and/or function, and can represent the influence, toxic action, virus infection etc. of influence as inborn error, morbid state, wound.Especially consider the hepatic diseases listed in this explanation.Use according to cell of the present invention and can in object, cause reconstructed tissue or regeneration.Allowing them to transplant or to move to the mode dosed cells in desirable tissue site, and the zone of reconstruct or refresh function defective.
Another aspect of the present invention is the method that prevents and/or treats hepatic diseases, comprise to the object of this treatment of needs especially people and use adult hepatic progenitor cell of the present invention or stem cell, the clone that contains described dirty progenitor cell or stem cell or cell mass (will be said especially, include but not limited to, LMBP6452CB is) or its offspring, the offspring who comprises differentiation, especially liver cell or liver cell like cell, randomly it is by genetic modification.Normally use, the part of expectation or the amount of systemic effect or performance promptly are provided usually with the treatment significant quantity.
Another aspect, the present invention relates to a kind of pharmaceutical composition, it comprises adult hepatic progenitor cell of the present invention or stem cell, the clone that contains described dirty progenitor cell or stem cell or cell mass, and (what will say especially is, include but not limited to, LMBP 6452CB is) or its offspring (comprise and break up the offspring), especially liver cell or liver cell like cell are randomly as above by genetic modification.
For example, rather than restriction, can be advantageously by injection (also comprise conduit use) or implant, as local injection, systematicness injection, intrasplenic injection (referring to Gupta etc., Seminars in LiverDisease 12:321,1992), introportal infusion, liver pulp injection, as under the Glisson's capsule, abdominal injection or intrauterine injection is gone into the embryo or fetus is used isolating hepatic progenitor cell of the present invention or stem cell, contain as described in the clone of dirty progenitor cell or stem cell or cell mass (what will say especially is, include but not limited to LMBP 6452CB system) or its offspring.
In a preferred embodiment, (what will say especially is can the present invention to be derived from the progenitor cell or the stem cell of liver, the clone that contains described hepatic progenitor cell or stem cell or cell mass by hepatocyte transplantation (LCT), include but not limited to LMBP 6452CB system) or its offspring (preferably by genetic modification) be used for organizational project and cell therapy.Liver cell is transplanted and liver stem cells transplanting (LSCT) is meant the mode that causes entering liver and cell implantation with any, preferably through portal vein, also can inject, or, inject the ripe liver cell of cell of the present invention or the technology of hepatic progenitor cell of comprising by intrasplenic injection by direct liver.
For example, after after separating or very low temperature were preserved and thawed, cell can provide in the form of the cell suspension in any substratum, preferably contains human albumin.
In one embodiment, the present invention considers to use patient's self hepatic tissue to separate progenitor cell of the present invention or stem cell.This cell be the patient from body, and use to the patient easily.In addition, if the patient has the hereditary defect that indicates the particular pathologies condition, can avoid this defective by the cell that genetic manipulation obtained.
In another embodiment, progenitor cell of the present invention or stem cell can never be to separate in patient's self the tissue to obtain.When considering when the patient uses this cell, preferably, select the liver organization of the inventive method processing and obtain progenitor cell or stem cell, (at least in the scope that can realize) increases the histocompatibility between patient and dosed cells to greatest extent, thereby the chance that the cell that minimizing is used is repelled by patient's immunity system (as, the transplant rejection that the host is right).
The ability that immunity system is distinguished oneself and nonego depends on the product of major histocompatibility complex (MHC) to a great extent, and its gene is on No. 6 karyomit(e) and belong to immunoglobulin gene superfamily.I class MHC product is made up of HLA-A, HLA-B and HLA-C; It is widely distributed and appear at basically on all karyocytes and hematoblastic surface.II class MHC product is made up of HLA-D, HLA-DR, HLA-DP and HLA-DQ; Its distribution is limited, comprises B cell, scavenger cell, dendritic cell, Langerhans cell and activatory (but not tranquillization) T cell.
The HLA gene locus is multiple alleles normally, as, use at least 26 HLA-A allelotrope of specific antibody identification, 59 HLA-B allelotrope, 10 HLA-C allelotrope, 26 HLA-D allelotrope, 22 HLA-DR allelotrope, 9 HLA-DQ allelotrope and 6 HLA-DP allelotrope.Because the HLA gene locus is closely linked, HLA antigen also can be that the haplotype form of guarding exists.
Need cell therapy of the present invention object can by at whether exist anti--hla antibody and HLA genotype thereof and/or phenotype (as, on the lymphocyte; As use serological method or hereditary DNA analysis) screen.Progenitor cell that obtains according to the present invention or stem cell or liver tissue source or its donor are being tested suitable tissue or the cell that its HLA phenotype and/or genotype and selection are used to use in typical case, it has the HLA haplotype consistent with the patient, or have most HLA antigen allelotrope common among the patient, and do not have or minimum HLA antigen at the anti--hla antibody that is pre-existing among the patient.The probability that transplanted cells is successfully accepted will improve along with the increase of identical HLA antigen number.Those skilled in the art will know that other variations of these considerations.
It is also conceivable that other approach that obtained to represent patient MHC spectrum, as the progenitor cell of the present invention's acquisition or stem cell or its offspring's genetic manipulation.
If cell is from external source (being non-from body), use the immunosuppressant therapy of following usually, as using immunosuppressor, (FK506) as ciclosporin or tacrolimus (tacrolimus).As an alternative, described cell can be wrapped in the permission fluid exchange but stop in the capsule of cell/cells contacting.The transplanting of microencapsulated cell is well known in the art, as Balladur etc., 1995, Surgery117:189-194; With Dixit etc., 1992, Cell Transplantation 1:275-279.Preferably, cell is from body, or the coupling of performance HLA as described is approaching.
In a further preferred embodiment, adult hepatic cell or its part be from non-human animal's object, preferred inhuman mammalian object.Can be in the member of identical, relevant or other non-human animals or non-human mammal species easily will be according to the present invention from non-human animal's preferred non-human mammalian subjects adult hepatic progenitor cell or stem cell or substitute thereafter in as research and liver disease therapy (as, contain xenotransplant, the bioartificial livers device of non-human animal or non-human mammal cell).For example, rather than restriction, being used for the particularly suitable non-human mammal cell of human treatment can be from pig.
A problem of the therepic use of relevant progenitor cell of the present invention or stem cell is the cell concentration that will reach best effect.The dosage of using can change, and can comprise following using first of subsequent applications; And can determine by disclosure one of ordinary skill in the art.In typical case, application dosage or dosage will provide the cell of treatment significant quantity, promptly reach local and the systemic effect and the performance of expection.
At present from the myelocytic human research of the single bony nodule of body, experience dosage from 1 to 4 * 10 7Individual cell does not wait, and has obtained challenging achievement.Yet different situations may need to optimize the amount of dosed cells.Therefore, the cell of using is different and different with the object of handling.In a preferred embodiment, 10 2To 10 9Between or 10 3To 10 9Between or 10 4To 10 9Between, as 10 4To 10 8Between or 10 5To 10 7Between, according to appointment 1 * 10 5, about 5 * 10 5, about 1 * 10 6, about 5 * 10 6, about 1 * 10 7, or about 2 * 10 7, about 3 * 10 7, about 4 * 10 7, about 5 * 10 7, about 6 * 10 7, about 7 * 10 7, about 8 * 10 7, about 9 * 10 7, or about 1 * 10 8Individual cell can be applied to human subjects.Yet, determine that accurately the treatment effective dose can be based on each patient's individual factors, comprise the time length after its stature, age, tissue injury size and damage take place, can determine easily according to the disclosure and this area general knowledge by those skilled in the art.
Preferably, being used to the cell mass purity that comprises progenitor cell of the present invention or stem cell used can be between about 50 to about 55%, between about 55 to about 60%, between about 65 to about 70%.More preferably purity can be between about 70 to about 75%, between about 75 to about 80%, between about 80 to about 85%; Most preferably purity can be between about 85 to about 90%, between about 90 to about 95%, between about 95 to about 100%; Can be according to stem cell purity as described in composing as cell surface marker in the cell mass to determine.Dosage can easily be adjusted (as needing to increase dosage than low-purity) by those skilled in the art.
Those skilled in the art can easily determine cell concentration in the composition to be administered and optional additive, vehicle and/or carrier in the inventive method.In typical case, any additives (except active progenitor cell or stem cell and/or cytokine) can 0.001 to 50% amount (w/w or w/v) be present in the solution of phosphate buffered saline buffer, in typical case, activeconstituents can be in microgram to the milligram rank, 0.0001 to about 5% (w/w or w/v) according to appointment, and preferred about 0.0001 to about 1%, and most preferably from about 0.0001 to about 0.05% or about 0.001 to about 20%, preferred about 0.01 to about 10%, and most preferably from about 0.05 to about 5%.
When using therapeutic composition of the present invention, be mixed with usually the unitary dose injection form (as, solution, suspension, dispersion agent, emulsion).The pharmaceutical dosage form that is suitable for injecting comprises aseptic aqueous solution and dispersion agent.As used herein, solution or dispersion agent comprise cell of the present invention and still keep active pharmaceutically acceptable carrier or thinner therein.Carrier can be acceptable solvent or the dispersion medium that comprises water for example, salt solution, phosphate buffered saline buffer, polyvalent alcohol (for example, glycerine, propylene glycol, liquid macrogol etc.) and suitable its mixture.
In addition, the various additives that can improve composition stable, sterility and isotonicity be can add, antibiotic antiseptic, antioxidant, sequestrant and damping fluid comprised.By various antibiotic and anti-mycotic agents, for example, parabens, butylene-chlorohydrin, phenol, Sorbic Acid wait and guarantee to prevent microbial activities.
Under many circumstances, wish that containing isotonic agent guarantees cell activity, for example, sugar, sodium-chlor etc.The desirable isotonicity of the present composition can realize by using sodium-chlor or other pharmaceutically acceptable reagent, as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.Especially preferred sodium-chlor for the damping fluid that contains sodium ion.
When the object that demand is arranged is introduced the offspring of target progenitor cell or stem cell or differentiation,, possibly described cell is mixed in biopolymer or the synthetic polymer in order effectively to increase the survival of cell.The example that is fit to biopolymer includes, but not limited to fibronectin, scleroproein, Fibrinogen, zymoplasm, collagen protein, proteoglycan.Its construction can comprise or not comprise the building of cytokine, somatomedin, differentiation factor or expression of nucleic acid etc.This biopolymer, as, can be the form that suspension or cell embed three dimensional gel wherein.This polymkeric substance is preferably biodegradable.
Can be by the absorption of using the slowly-releasing absorption agent to prolong injectable medicine type, for example, aluminum monostearate and gelatin.According to the present invention, yet, the carrier of any use, thinner or additive all necessary and progenitor cell or stem cell compatibility.
Can be by implementing to prepare aseptic injectable solution in cell used when of the present invention mixes aequum with other compositions (needs) of various amounts the suitable solvent.
This composition also can be mixed with suitable carriers, thinner or vehicle, as sterilized water, physiological saline, glucose, dextrose etc.Composition can comprise auxiliary substance, and as wetting agent or emulsifying agent, pH buffer reagent, jelling agent or viscosity enhancement additive, sanitas, seasonings, pigment etc., this depends on route of administration and preparation requirement.
Can the reference standard text, as " Remington ' s pharmaceutical science " the 17th edition, 1985 by with reference to incorporating into herein, does suitable preparation, and avoid unnecessary experiment.
If necessary, can use pharmaceutically acceptable thickening material that the viscosity of composition is maintained selected level.The preferable methyl Mierocrystalline cellulose is because it is easy to commercial acquisition and is convenient to operation.Other suitable thickening comprise, for example, and xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, carbomer etc.The concentration of preferred thickening depends on selected reagent.When using certain amount, under this amount, will obtain selected viscosity.Usually prepare viscous composition by in solution, adding this thickening material.
Other purposes of progenitor cell of the present invention or stem cell
Considered progenitor cell of the present invention or stem cell below, (what will say especially is to contain the clone of described dirty progenitor cell or stem cell or cell mass, include but not limited to, LMBP 6452CB is) or its offspring (offspring who comprises differentiation, especially liver cell or liver cell like cell, the randomly offspring of its genetic modification) other purposes.
Therefore, progenitor cell or stem cell or its differentiation derivative, especially liver cell or liver cell like cell, can be used for detecting cell response (as toxicity) to bioactive agents (biological agent or pharmacologic agent), comprise that the derivative that makes cell culture or its differentiation contacts one or more and plants biology or pharmacologic agent, evaluation is planted one or more kind cell response, the relatively cell response of cell culture and cell responses of control cultures of biology or pharmacologic agent to one or more.This reaction can be determined by detection molecules is active, for example, but be not limited to the enzyme of alkaline phosphatase, Cytochrome P450, urea approach etc.
In addition, for example, can be by offspring, especially liver cell or the liver cell like cell of progenitor cell of the present invention or stem cell or its differentiation, screen cytokine, chemokine, pharmaceutical composition and somatomedin, more clearly to illustrate it to the differentiation of this cell and the effect of function.
The present invention has also designed a kind of engineered organ, or its part, or specific part, comprise the destination organization and the tissue engineering devices of cytokine, somatomedin or differentiation factor randomly, cell wherein of the present invention is used for producing tissue, especially liver organization especially comprises hepatocellular tissue.Engineered organ can cooperate biocompatible scaffold to use, and described support sustenticular cell is with three-dimensional conformation (structure) growth, and support is biodegradable.From the implantable object that needs to change organ, its part or specific part of the engineered organ of stem cell generation of the present invention.The present invention has also designed the purposes of the cell of stem cell or its differentiation as bio-reactor (as the liver supplementary unit).
The host can be implanted in organ, its part or zone from stem cell of the present invention.Transplanting can be from body, and promptly the donor from organ or the unitary stem cell of organ is the acceptor of engineering tissue.Transplanting can be allogenic, and promptly the donor from organ or the unitary stem cell of organ is not the acceptor of engineering tissue.In case be transferred to the host, engineered organ can reappear the function and the structure of natural host tissue.Engineered organ will help the multiple application of theme of the present invention, comprise treatment cancer and other diseases, birth defect or because the damage that surgical excision causes disclosed herein.
As the instrument of drug abuse test and performance history, liver cell and offspring thereof can be used for assessing and treat that the gene expression pattern that developing drugs causes changes.The gene expression pattern that potential drug causes changes and can compare with the drug-induced variation of those known effect livers.This will make drugmaker screen the influence of liver in early days in exploitation compound, thereby save time and money.The whole pedigree of liver cell from the progenitor cell to the mature cell, also can be used for testing drug to the toxicity of liver, the how metabolism of research medicine.At present, drugmaker has any problem in the conforming liver cell supply side that is used for toxotest of acquisition.The inventive method and cell have satisfied this demand.
In addition, adult hepatic progenitor cell of the present invention or stem cell or its offspring (comprise and break up the offspring), especially liver cell or liver cell like cell can be used as the biological components of detoxification device, as liver perfusion or liver supplementary unit.
Traditional liver supplementary unit comprises a firm plastic casing and hollow semi-permeable membrane fibers, and wherein inoculation has the liver cell of stem cell or differentiation or from stem cell.This fiber can use collagen, lectin, ln or fibronectin to handle, and is used for adherent cell or stays undressed.Come detoxification according to known program by device perfusion body fluid, and then fail back the patient.The example of the LAD of a suitable cell of the present invention has been described among the open PCT US00/15524 of international monopoly.
Adult hepatic progenitor cell of the present invention or stem cell or its offspring can substitute ripe liver cell in vitro differentiation and in " ADMET " (use, distribution, metabolism, elimination and toxicology) or cytotoxicity test.
The liver cell or the liver cell like cell that obtain by adult hepatic progenitor cell of the present invention or stem cell or its offspring differentiation can provide a kind of be used to study liver growth, liver cell metabolism or the biological external model of liver cell; Be used to screen differentiation, propagation or toxicity molecule.Also consider the genetic manipulation of this cell, they can be used to study liver growth, liver cell metabolism or the relevant gene of biology.
To the present invention be described by following embodiment now, the scope that this does not limit the present invention in any way.
Embodiment
The liver cell separation method
From partly obtaining the human liver cell from healthy corpse or the whole liver or the liver that do not have a heartbeat donor.After total blocking-up time (as, after the time of blocking-up always 6 to 12 hours) isolated cell, before perfusion liver all in University of Wisconsin's medium (the University of Wisconsinmedium) be stored on ice.Use classical two to go on foot perfusion technique isolating hepatocytes { Seglen, 1976}{Stephenne, 2005}.(the Earl balanced salt solution does not contain Ca to liver organization with EGTA solution via tangible blood vessel subsequently ++And Mg ++, 0.5mM EGTA, 5mM Hepes, 2mg/l gentamicin and 100, the 000IU/I penicillin G) and digestive ferment solution under 37 ℃, poured into separately 9 to 12 minutes.(EBSS contains Ca to digestion solution ++And Mg ++, 5mM Hepes, 2mg/l gentamicin and 100, the 000IU/I penicillin G) contain the Trypsin inhibitor SBTI of 0.9 mg/ml collagenase P and 0.03 mg/ml.Cutting Glisson's capsule and slight vibration discharge liver cell.Stop digestion (M199 substratum, 5mM Hepes, 2mg/l gentamicin and 100,000IU/I penicillin G) with the ice-cold cleaning medium that contains 0.03 mg/ml Trypsin inhibitor SBTI and 100 milliliters/rise human plasma.Filter and the cleaning cell by four metallic sieves, mesh size is respectively 4.5mm, 1mm, 0.5mm and 0.25mm.By under 1200rpm centrifugal 3 minutes, with ice-cold M199 cleaning medium washed cell 3 times.
Primary cell culture
With single cell suspension be resuspended in be supplemented with 10% foetal calf serum (FCS) (Perbio, Hyclone), in the Williams ' E matrix of 25ng/ml EGF (Peprotech), 10 μ g/ml Regular Insulin, 1 μ m dexamethasone and 1% penicillin/streptomycin (P/S) (Invitrogen company) (Invitrogen company).Cell inoculation is in the shaking on bottle or the plate of I type mouse tail collagen (BD Biosciences) bag quilt (as, 6 orifice plates) (Greiner Bio-one), and containing 5%CO 2Cultivate down at 37 ℃ in the water saturated environment.After 24 hours, change substratum and remove not adherent cell, upgraded in after this per 3 days.In fortnight, microscopic examination every day culture, per three days analysis substratum.Then substratum is changed into have high glucose concn be supplemented with 10%FCS (Perbio, Hyclone) and the DMEM (Invitrogen) of 1%P/S (Invitrogen) so that quicken the hepatocellular removal of adult.Confirm to have spontaneous subsequently appearance of cell type of mesenchyme sample form through phase microscope, breed and fill up the blank space of orifice plate.Cultivate between 15 to 20 days and these cells occur, and present flat pattern, obvious tenuigenin and/or the former forming core of ovum (Fig. 1) of one or two kernel is arranged.When reaching 70% when converging, peel off cell with 0.25% trypsinase and 1mM EDTA, and with the renewed vaccination of expectation concentration.The cell suspension analysis revealed that carries out with the flow cytometer cell mass homogenization that becomes of going down to posterity after 2 times.For go down to posterity at every turn, also can come the analysis of cells suspension with RT-PCR and immunofluorescence.
Cell characteristics
For fear of being carried out the phenotype that immunocytochemistry is studied these cells by the other types cell contamination.Because they derive from liver, have analyzed specificity marker thing such as albuminous expression in Paraformaldehyde 96 fixed cell.As shown in Figure 2, the albumin of using mono-clonal (Sigma clones HAS-111) or polyclone (Chemicon) antibody test in liver cell, to express only.Also estimated the expression of mesenchymal cell mark abreast, shown that these cells are vimentin and α smooth muscle actin immunity positive (Fig. 2).Its phenotypic characteristic has very big stability in 7 times the research of going down to posterity up to now.
Cytodifferentiation:
With 0.5-1 * 10 4/ cm 2Density cell inoculation is coated with in the DMEM that is supplemented with FCS and P/S on 6 orifice plates of I type mouse tail glue primordial covering.Change substratum into Iscove ' smodified Dulbecco ' s substratum (IMDM) (Invitrogen) after 24 hours.For inducing, cell was hatched for 2 weeks in the inducing culture that contains IMDM additional 20ng/ml HGF (Biosource), 10ng/ml bFGF (Peprotech) and 0.61g/l niacinamide (Sigma).After this, cell is hatched in containing the additional 20ng/ml oncostatin M (Sigma) of IMDM, 1 μ M dexamethasone (Sigma), 50mg/ml ITS (Regular Insulin, Transferrins,iron complexes, selenium) maturation medium (Invitrogen).For inducing and maturing step, changed in per 3 days and the analysis substratum.After above mixture (cocktail) contacted, cell began to lose its sharpened edge, shrink gradually and lose its initial form and present polygonal shape (Fig. 3).
Flow cytometry
With 1200rpm collecting cell after centrifugal 5 minutes, and cell is resuspended among the PBS with the concentration of 500 to 1000 cell/μ l.Under 4 ℃, hatched 30 minutes with antibody then.Corresponding control antibodies hypotype is used to estimate the non-specific binding of monoclonal antibody.The rinsing cell is resuspended in then
Figure A20068004882200591
(Beckham Coulter) reads with Beckham Coulter flow cytometer.
RT-PCR
According to the specification sheets of manufacturers, use and extract total RNA the cell of TriPure separation agent (Poche) on growing in 6 orifice plates, produce cDNA with the reverse transcription test kit.Carry out pcr amplification with polysaccharase prolongation enzyme and suitable primer in the 25 μ l final volume.After this, sample carries out electrophoresis and observes nucleic acid by ethidium bromide staining on 1% sepharose.
Immunofluorescence
Immunostaining, at room temperature, with the cell of 4% Paraformaldehyde 96 (V/V) fixed growth on the 12mm circular lid glass sheet of I type mouse tail collagen-Bao quilt 15 minutes, the 1%TBS solution (V/V) of after this using Triton X 100 was with cell permeabilization (Permeabilized) 15 minutes (Tris-HCl 50mM, NaCl 150mM, pH 7.4).Prevented non-specific immunity dyeing in 1 hour by in containing the TBS solution of 3% skim-milk, hatching under 37 ℃.Same subsequently anti-at room temperature the hatching in same solution 1 hour of cell used TBS rinsing 5 times, resists with two and hatches 1 hour (1/500) together.With nuclear dyestuff DAPI (1/5000) nuclear is carried out dyeing in 30 minutes.After the rinsing 3 times, place Fluoprep medium (BioMerieux, Brussels, Belgium) also with Olympus 1 * 70 inverted microscope inspection of being furnished with CCD camera (T.I.L.L.photonics, Martin's Si Reed, Germany) prepared product.With the xenon lamp of being furnished with monochromator (monochromator) (T.I.L.L.photonics, Martin's Si Reed, Germany) obtain exciting light (for Cy-3, FITC and DAPI be respectively 552,488 and 372nm).Use suitable filter and be used TILLvision software and obtain digital image.
The Molecular Detection of progenitor cell of the present invention or stem cell
Use aforesaid method,, set up the express spectra (ADHLSC) of following various kinds of cell mark in the experiment that is used for setting up cell of this embodiment:
The CD90 positive.CD90 or Thy-1, a kind of cell surface protein is regarded as the sign that mesenchyme is.
The CD44 positive.CD44 is a kind of cell adhesion molecule and is used for discerning the interstital stem cell of some type (MSC) at least.
The vimentin positive.Vimentin is often can detected III type median fiber in a kind of mesenchymal cell and the inoblast.
The albumin positive.Albumin is a kind of plasma proteins that is produced justacrine by liver.In liver cell, find that usually albumin is a kind of cytoplasm protein.
The CD29 positive.CD29 is also referred to as and integrates plain β-1, is a kind of transmembrane glycoprotein, also is present in the hepatic tissue, is considered to form participation and the interactional functional receptor mixture of extracellular matrix together with integrin alpha.
The CD73 positive.CD73 is a kind of 5 ' of a kind of mesenchyme mark-circumscribed phosphonuclease that is considered to.
The CD49b positive.CD49b is also referred to as integrin alpha-2 or collagen protein acceptor, and the interaction of participation and extracellular matrix.
The HLA-ABC positive.HLA-ABC (human leucocyte antigen A, B and C) is that I class major histocompatibility complex antigen forms the film heterodimer.
The alpha-fetoprotein of low expression level.Alpha-fetoprotein is to express, reflect the protein of entoderm pedigree in a kind of primitive endoderm (primitiveendoderm) growth course and the whole maturation.The high level expression alpha-fetoprotein discloses tumorigenicity usually and separates (tumorigenic shunt).
α-1 antitrypsin the positive.α-1 antitrypsin is a kind of liver synthetic plasma proteins.
Glucose 6-Phosphoric acid esterase (G6P) positive.G6P a kind ofly becomes the liver enzyme of glucose and inorganic phosphorus with glucose 6-phosphoric acid hydrolysis, makes blood sugar enter blood from liver.
Cytochrome P450 1B1 (CYP1B1) positive.CYP1B1 is the derivable cytopigment of a kind of Dioxins, and it is responsible for the I phase metabolism of matrix various on the far-ranging structure.
Cytochrome P450 3A4 (CYP3A4) positive.CYP3A4 is the metabolic key enzyme of a kind of participation heterobiotin.
Hepatocyte neclear factor 4 (HNF-4) positive.HNF4, a kind of nuclear receptor is a kind of transcription factor that energy metabolism is regulated that participates in.
Tryptophane 2,3-dioxygenase (TDO) positive.TDO is first enzyme that participates in tryptophane oxidation in liver.
Tyrosine aminotransferase (TAT) positive.TAT is a kind of participation amino acid metabolism and gluconeogenetic plastosome liver specificity enzyme.
Glutamine synthetase (GS) positive.GS is a kind of key enzyme of ammonia assimilation.
Gamma-glutamyltranspeptidase (GGT) positive.GGT is a kind of enzyme that participates in glutathione metabolism.
Cytokeratin 8 (CK8) positive.CK8 is the specific median fiber of a kind of epithelial cell.
Multidrug-resistance protein 2 (MRP2) positive.MRP2 is a kind of organic anion translocator of being responsible for organic anion in the cell is exported to from liver cell biliary tract.
Glutamate transporter 2 (EAAT2) positive.
The above-mentioned liver function and metabolic minute subrepresentation ADHLSC clone and liver phenotype of much may participating in occur close ties are arranged.
The CD117 feminine gender, CD117 is also referred to as c-kit, is the cell surface receptor of discerning HSC and MSC on a kind of medullary cell type, thereby characterizes undifferentiated to a great extent stem cell.
The CD34 feminine gender.CD34 is the cell surface protein on a kind of medullary cell, indicates HSC and endothelial progenitor cells.
The CD45 feminine gender.CD45 is also referred to as human leucocyte antigen, is the tyrosine phosphatase that a kind of hematopoietic lineage cell (comprising hemopoietic stem cell) is expressed.
The CD105 feminine gender.CD105 is also referred to as SH2 or endoglin, is a kind of adhesion molecule.It also is considered to a kind of mescenchymal stem cell mark.
The CD133 feminine gender.CD133 is a kind of hemopoietic stem cell mark.
The HLA-DR feminine gender.These II class major histocompatibility complex antigens are film heterodimers of the restricted expression of antigen presenting cell.
The Oct-4 feminine gender.Oct-4 is a kind of transcription factor of only being expressed by multipotential stem cell, and is that to keep undifferentiated state necessary.
Cytokeratin 19 (CK19) feminine gender.CK19 is widely used as the mark that the biliary tract cell is a bile duct cell.
Cytochrome P 2B6 (CYP2B6) feminine gender.In CYP2B6 participates in-and the heterobiotin metabolism.
CD54: intercellular adhesion molecule-1 (ICAM-1), a kind of membrane glycoprotein.
Be not intended to limit by any way, the inventor has proposed the following explanation possible to above-mentioned data according to the knowledge of its pair cell mark: these mark combinations have defined a kind of clone originally, and it expresses mesenchyme pedigree mark (CD90, CD73, vimentin, CD44) and the distinctive mark of liver differentiation pathway (CD29 and albumin, α-1 antitrypsin, HNF4, MRP2 translocator).By immunofluorescence and RT-PCR detect exist albumin come down hard upon by stellate cell pollute may.As if ADHLSC neither liver stellate cell (the albumin positive) neither multipotency does not break up mescenchymal stem cell (CD45 feminine gender, CD34 feminine gender, CD117 feminine gender, Oct-4 feminine gender).ADHLSC pedigree consistent with the liver pedigree (the CD29 positive, express albumin and α-1 antitrypsin) but do not express typical biliary tract mark (CK19 and 7 feminine genders).Therefore, cell of the present invention and clone can be characterized as being the mescenchymal stem cell system with liver cell progenitor cell feature with a kind of but be not the mode of restriction.
The transplanting of uPA-SCID mouse, histology and immunohistochemistry
1,000,000 ADHLSC (〉=90% activity) are expelled to 6-to 14 age in days uPA + /+The spleen of-SCID mouse.Before the transplanting, mouse does not show detectable serum albumin.The immunohistochemical analysis of carrying out the mouse liver sample on phenodin and the thick liver section of the painted 4 μ m-of eosin (HE) is used for the histopathologic overall evaluation.For immunostaining, under the room temperature with first antibody with the liver section overnight incubation.Detect after hatching section with the polymkeric substance of peroxidase labelling and substrate chromogenic reagent (the Envision-DAB system, Dako, Carpinteria, CA).Use haematoxylin redyeing.
The transgene mouse model that is used for this purpose combines with the hepatic pathology with immunodeficient disease (SCID) (uPA).After the spleen of ADHLSC suspension is transplanted, allow uPA + /+-SCID mouse recovered for 10 week.Transplanting has the analysis of the uPA/SCID mouse liver of ADHLSC to represent that these cells can transplant (Fig. 4) and be divided into ripe liver cell (Fig. 5).In addition, detect human serum albumin in the transplanting mice serum after these transplanted for 10 weeks, the alpha-fetoprotein level of expression (mark that tumour is educated) still detect less than.
The cell of transplanting shown in microscopic examination does not have hyper-proliferative, and expression does not have the expression level of tumorigenesis colony and tumorigenesis mark alpha-fetoprotein and Ki67 normal.The normal expression level is equivalent to the expression level measured in the normal liver cell basically, and is lower than the expression level of people's liver cell system as measuring among the HepG2 of tumorigenicity transformation.
Embodiment 2
One is derived from the progenitor cell or the stem cell of liver, the clone that comprises described progenitor cell or stem cell and/or cell mass with the present invention (what especially will say is, include but not limited to LMBP 6452CB system) or its offspring's (randomly by genetic modification) exemplary process can be as follows.
Inject cell by continuous injection, preferably be no more than 25 to 50 * 10 6Individual cell/kg, preferred interval 4 hours, or 8 hours at interval, or surpass 8 hours until 1 week, or surpassed for 1 week.Continue a few days injection 250 * 10 6Individual cell/kg, or 500 * 10 6Total cell concentration of individual cell/kg, a preferred week or preferred two weeks.The words that need repeat the series injection, every other month or every six months or every 1 year or longer.
Radioactivity and or ultrasonic guidance under, through puncture needle or through subcutaneous catheter through Port-a-cath R device or through Broviac R device via surgery be inserted into any drainage to pylic blood vessel (preferred inferior mesenteric vein or colic vein) by direct puncture access door vein.Conduit can be retained a few hours in position, and preferred a couple of days, preferably several weeks, or preferred several months were until 2 years, or the preferred longer time is so that the time of needs repetition infusion.
Immunosuppression starts from infusion same day, preferred the day before yesterday, preferably uses tacrolimus (FK506) and steroid.The preferred initial period 8ng/ml of the blood levels of tacrolimus, 6ng/ml after 3 months, 4ng/ml after 6 months remains on about 4ng/ml then.Steroid preferably gives prednisone or Ultracortene-H, and the preferred initial period is at the 1st day 5mg/kg, at the 2nd day 4mg/kg, at the 3rd day 3mg/kg, at the 4th day 2mg/kg, at the 5th day 1mg/kg, in the time of 3 months, gradually reduce 0.25mg/kg then, and in the time of 6 months, stop.The alternate immunosuppression can comprise, separately or associating (using), the polyclone of ciclosporin A, anti-IL2 receptor antibody, antithymocyte globulin or any anti-human lymphocyte or monoclonal antibody, mycophenlate mofetil mofetyl or azathioprine or any antimetabolite, ciclosporin, rapamycin (rapamune) or any other neurocalcin (calcineurin) inhibitor.

Claims (39)

1. derive from the isolating progenitor cell or the stem cell of the liver of growing up, it is characterized in that its coexpression liver cell marker albumin (ALB) and at least one mesenchyme mark, preferred one, more than one or whole mark CD90, CD73, CD44, vimentin and α-smooth muscle actin (ASMA).
2. the isolating progenitor cell or the stem cell of claim 1, it also expresses one or more other liver or the liver cell mark that is selected from CD29, alpha-fetoprotein (AFP), α-1 antitrypsin, HNF-4 and the MRP2 translocator.
3. each isolating progenitor cell or stem cell in the claim 1 or 2, its coexpression liver cell mark albumin (ALB), at least one liver landmarks thing CD29 or alpha-fetoprotein (AFP) and at least one mesenchyme mark, preferred one, more than one or whole mark CD90, CD73, CD44, vimentin and α-smooth muscle actin (ASMA).
4. the isolating progenitor cell or the stem cell of claim 3, it also expresses one or more liver cell mark α-1 antitrypsin or MRP2 translocator.
5. each isolating progenitor cell or stem cell in the claim 1 to 4, it is positive to CD90, CD29 and CD44, randomly also CD73 is positive, and is the albumin positive, the vimentin positive and α-smooth muscle actin positive.
6. each isolating progenitor cell or stem cell in the claim 1 or 2, wherein said cell is negative to CD45, CD34, CD117 at least, preferably Oct-4 also is negative, and CK-19 is negative.
7. each isolating progenitor cell or stem cell in the claim 1 to 6, wherein said cell has mesenchyme sample form, preferably include following each or all: monolayer growth, flat pattern, wide tenuigenin and avette nucleus with one or two kernel.
8. each isolating progenitor cell or stem cell in the claim 1 to 7, wherein said cell can be divided into liver cell or liver cell like cell, and is not divided into the mesoblastema type.
9. clone or cell mass, it comprises each described isolating hepatic progenitor cell or stem cell in the claim 1 to 8, and its offspring.
10. the progenitor cell of each hepatic progenitor cell or stem cell or claim 9 or stem cell line or cell mass in the claim 1 to 8, wherein said progenitor cell or stem cell are the people.
11. isolating people's adult hepatic progenitor cell or stem cell and clone, be deposited in Belgian microorganism cooperation preservation center (BCCM) on February 20th, 2006 according to budapest treaty, its preserving number is LMBP6452CB, its subbreed, comprise clone's subbreed, and the offspring.
12. obtain isolating progenitor cell or stem cell or comprise the method for the cell mass of described progenitor cell or stem cell, this method comprises: (a) dissociate adult hepatic or its part, to form the primary cell group from described adult hepatic or its part, (b) with described primary cell group bed board to cell can adherent substrate on, (c) will continue to cultivate preferably at least 10 days, at least 13 days or at least 15 days at least 7 days from the described suprabasil cell of adhering to of this primary cell group.
13. the method for claim 12, wherein step (c) afterwards with described passage at least once, preferably at least twice.
14, isolating adult hepatic progenitor cell or stem cell, its clone and/or comprise the cell mass of described cell, it can obtain or directly be obtained by described method with each method in the claim 12 or 13.
15. the isolating adult hepatic progenitor cell of claim 14 or stem cell, its clone and/or comprise the cell mass of described cell, wherein said method comprises: (a) from object dissociate, preferably by two step collagenase method dissociate adult hepatic or its parts, described object preferably vertebrates, Mammals, be more preferably people's object, to form the primary cell group from described adult hepatic or its part; (b) with the bag of described primary cell group bed board in the Williams Medium E substratum by in the substrate of type i collagen, described substratum comprises foetal calf serum (preferred 10% (v/v)), EGF (preferred 25ng/ml), Regular Insulin (preferred 10 μ g/ml) and dexamethasone (preferred 1 μ M); (c) continue to make in 24 hours cell adhesion from described primary cell group to described substrate, substratum is replaced by has fresh culture of forming in (b) subsequently; (d) in two time-of-weeks (preferred 15 days) in substratum described in (c) culturing cell; (e) substratum is replaced by the DMEM that comprises high glucose and FCS (preferred 10%), continues culturing cell, progenitor cell of the present invention thus or stem cell occur and propagation; (f) randomly and preferably, make cell reach about 70% and converge,, cultivate in the substrate of wherein said cell bed board in (b) and in the substratum in (e) passage at least once and preferably at least twice.
16. a composition, it comprises in the claim 1 to 8,10 or 11,14,15 each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15.
17. the composition of claim 16, wherein said liver cell are people's liver cell or mammalian liver cell.
18. the method for treatment hepatic diseases comprises in the claim 1 to 8,10 or 11,14,15 of using significant quantity each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15.
19. each described clone or cell mass or its offspring in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15 in the claim 1 to 8,10 or 11,14,15, comprise and break up the offspring, preferred liver cell or liver cell like cell, described cell is used for the treatment of.
20. each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15 in the claim 1 to 8,10 or 11,14,15, or its offspring, comprise and break up the offspring, preferred liver cell or liver cell like cell are used for the treatment of purposes in the medicine of hepatic diseases in preparation.
21. the method for claim 18 or the purposes of claim 20, wherein said hepatic diseases comprise pku and other amino acid metabolism diseases, hemophilia and other thrombin defectives, familial hypercholesterolemia and other lipid metabolism disorders, urea cycle disorder, glycogenosis, galactosemia, fructosemia, tyrosinemia, protein and carbohydrate metabolism defective, organic aciduria, mitochondrial disease, peroxysome and lysosome are not normal, protein synthesis is unusual, liver cell translocator defective, glycosylation defect, hepatitis, liver cirrhosis, congenitalDy olism, acute hepatic failure, acute liver infects, acute chemical toxicity, chronic liver failure, cholangitis, cholehepatocirrhosis, Alagille syndrome, alpha-1-amtitrypsin deficiency, autoimmune hepatitis, Biliary atresia, liver cancer, the hepatic pouch venereal disease becomes, fatty liver, galactosemia, gallbladdergallstonecholetithiasis, Gilbert syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, and other virus infection type hepatitis, porphyria, primary sclerosing cholangitis, the Reye Cotard, sarcoidosis, tyrosinemia, I type glycogen storage disease, or Wilson's disease.
22. a pharmaceutical composition, it comprises in the claim 1 to 8,10 or 11,14,15 each described clone or cell mass and pharmaceutically acceptable carrier in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15.
23. implement the method for in vitro toxicity test, it comprises:
Make in the test medicament contact claim 1 to 8,10 or 11,14,15 each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15, and observe of at least a influence of this test medicament if any described liver cell group.
24. the method for claim 23, wherein said at least a influence comprise pair cell survival, cell function or to the two influence.
25. implement the method for external drug metabolism study, it comprises: (i) make in the test medicament contact claim 1 to 8,10 or 11,14,15 each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15, (ii) if any, observe after the time and the relevant at least a variation of this test medicament at presumptive test.
26. the method for claim 25, wherein said at least a variation comprise that described test medicament is in structure, concentration or the variation of the two.
27. comprise the liver supplementary unit of pedestal, described pedestal holds in the claim 1 to 8,10 or 11,14,15 each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15.
28. therapeutic gene is expressed wrong method, it comprises: (i) with each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15 in the functional copy importing claim 1 to 8,10 or 11,14,15 of gene, transform colony to provide; (ii) described at least a portion that has transformed colony is imported patient's liver, wherein the functional copy of this this gene of needs of patients.
29. be used for the treatment of the composition of genetic expression mistake, it comprises in the claim 1 to 8,10 or 11,14,15 that has transformed each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11 that has transformed, 14,15, has wherein imported the functional copy of gene.
30. be used for the treatment of the pharmaceutical composition of genetic expression mistake, it comprises in the claim 1 to 8,10 or 11,14,15 each described clone or cell mass and pharmaceutically acceptable carrier in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15, has imported the functional copy of gene in the wherein said cell.
31. strengthen the method for damage or ill liver regeneration, it comprises to described liver uses in the claim 1 to 8,10 or 11,14,15 of significant quantity each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15.
32. test is used for the treatment of the method for the beneficial agents of liver infection, it comprises that (i) infects in the claim 1 to 8,10 or 11,14,15 each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15 with the target infectious agent, so that infected colony to be provided, (ii) make the test medicament of described infected population exposed predetermined amount, (iii) if any, observe this contact to the described influence of having infected colony.
33. the method for claim 32, wherein said infectious agent comprises microorganism.
34. the method for claim 32, wherein said infectious agent comprise one or more virus, bacterium, fungi or its combination.
35. the method for claim 32, wherein observed influence comprises the influence to the former virus replication of viral communication.
36. the method for claim 35, the former hepatitis virus that comprises of wherein said viral communication.
37. productive target method of protein, it comprises each described clone or cell mass in each described hepatic progenitor cell or stem cell or the claim 9 to 11,14,15 in the functional gene importing claim 1 to 8,10 or 11,14,15 of coding target protein, transcribing, translating and randomly cultivating described liver cell group and gather in the crops described target protein under the effective condition that takes place of posttranslational modification.
38. the method for claim 37, wherein said liver cell are people's liver cells.
39. the method for claim 38, wherein said target protein comprises vaccine antigen.
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