CN101356264B - Isolated liver stem cells - Google Patents

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

The liver stem cells separating

Technical field

The present invention relates to hepatic progenitor cell or stem cell from the separation of adult hepatic, and purposes in medical science, hepatopathy, liver metabolism inborn defect, transplanting, communicable disease, liver failure.The invention still further relates to the method, its culture, differentiation front and back sign and the purposes in animal model, artificial organ device, toxicology and the pharmacology of transplanting, human diseases thereof that separate these cells.

Background technology

Liver is vitals, a lot of important functions of its performance, as (xenobiotic) detoxification of the homeostasis of glucose, xenobiotic or macromole synthetic.Therefore, one of multiple liver function is impaired can make a significant impact health.According to the World Health Organization (WHO), the sickness rate of global urgency or chronic hepatopathy makes these diseases become the cause of death of the 5th to the 9th.So far, to late period hepatic diseases radical cure method only have hepatic transplantation.The patient's of experience surgery liver transplantation final result is fine, more than 95% rehabilitates.But even if there is the new surgical technology including liver segmentation and Living Donor of Living Liver Transplantation Eastern Hepatobiliary Hospital, the mortality ratio clamping in treatment list that makes day by day in short supply of organ constantly rises.Therefore, the important goal in transplantation medicine research proves the potential use of liver cell (liver cell) in liver regeneration and liver disease exactly.

It is a kind of new methods that liver cell is transplanted (liver cell transplantation, LCT), relates to infusion liver cell suspension in acceptor hepatic portal system.Be to recover acceptor liver function and the ill liver parenchyma (liverparenchyma) of reconstruction (repopulation) as transplanting its object of result.First LCT is proved in animal model effectively, has wherein shown that 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 exhaustion for the treatment of acute hepatic.These researchs impel clinical position, and person expands LCT for other indications, and so far, at least 30 cases (people 1997.Transplant Proc 29 (4) such as Strom: 2103-6) of various defects has been reported in the whole world.In the specific field of metabolic trouble, in 13 cases, report that liver cell is used for the treatment of I type Crigler-Najjar syndrome, ornithine cycle defect or orphan disease as the purposes of baby Refsum disease.The improvement that these studies have shown that the implantation of liver cell in liver parenchyma and reach subsequently 18 months status of patient after transplanting.

But, because the mature human's liver cell for transplanting is still limited, in fact more or less the same limited with full liver operability, research is also intended to obtain transplantable cell from other sources, as progenitor cell and stem cell from embryo or adult source, these cells for example can increase in vitro, and can especially after transplanting, in body, be divided into the mature hepatocytes of function.Therefore, the useful method in the relevant various diseases for the treatment of liver related disease or the patient's condition making new advances in the urgent need to exploitation, while especially considering the available treatment deficiency of these diseases of most.

In history, owing to observing dry (ES) cell of embryo and infinitely clone division and multipotency and be divided into the ability of the daughter cell (daughter cell) of whole tissue, it was once considered to only participate in organ and occurred.On the other hand, the regenerative process in adult organ is in typical case owing to adult progenitor cells.But, consider the stem cell of having found to express embryo's mark in adult organ, this theory is corrected.Therefore while, describing now stem cell and progenitor cell feature not only based on the also existence based on specific cell mark of growth course (embryo is to adult).In fact, cell sign thing is as the expression of membranin or transcription factor can along with differentiation pathway, great changes have taken place, and they have reflected various stimulations (as environmental stimulus) and cell requirements.Conventionally in atomization, observe stem cell and indicate the mark of its versatility as Oct-4 by stopping gradually showing, contribute to the mark of follow-up phase 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 on the other hand entoderm pedigree starts 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, due to the versatility of ES cell, expect its any organ of can regenerating.In fact, this area Zhong Zhetiao is extensively explored on road.But ES cell tends to cause tumor growth in the time that ES cell is introduced in any other tissue in addition of uterus (inutero).Therefore, in its body, apply because carcinogenic risk is still restricted.Even if ES cell breaks up successfully in vitro before, all safe not for mankind's inoculation.

Safer alternative is to use adult progenitor cells, and unlike ES cell, it tends to show the daughter cell of limited clone's splitting ability and the more limited destiny of differentiation generation.In liver, adult progenitor cells was described as elliptocyte (bile duct cell and liver cell precursor) or microhepatia cell like cell.But its rareness in normal adult organ makes its medical usage become difficulty.

Therefore, have reduce or do not depart to carcinogenic risk and demonstrate the adult stem cell of clone's splitting ability will represent the major progress in Transplanted cells source.Polytype adult stem cell is transplanted in research and is evaluated at liver cell at present.For example, become the more ability of mature cell owing to having from another pedigree transdifferentiation, the mescenchymal stem cell from periphery or Cord blood (mesenchymal stem cell, MSC) is studied.And, also aspect liver regeneration potential, the hemopoietic stem cell from marrow is being studied.

The vitro characterization of adult stem cell is difficulty still, the current 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) expression of the mark of reflection level of differentiation, thereby indicate to a certain extent and possible different offsprings (iii) broken up destiny in afterwards external or body.Therefore, characterize and distinguish the adult stem cell obtaining 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) differentiation mark (as there being albumin) and (iii) mark of at least one indication stem cell destiny.

According to current knowledge, liver is mainly derived from entoderm, and liver cell is a part of entoderm pedigree (lineage).But hepatocellular formation also relates to the interaction between entodermal epithelium and cardiogenic mesoderm.In addition,, in fetation, hematopoietic cell generates and occurs in liver.Consider developmental this influencing each other, in the time mentioning the mark existing in adult hepatic stem cell, need to have opening thought, because the mark of entoderm, mesoderm and/or hematopoiesis system all can be expected.

In the time of assessment level of differentiation and cell type ownership, can evaluate different cell sign things, as what carry out subsequently in the disclosure.For example, in cell differentiation procedure, some mark reduces or disappears, and other marks can increase or occur to also have some can be maintained to specialization and functional cell.According to limiting examples, between organogenetic period, be 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; And CK-19, albumin and alpha-fetoprotein CK-7).In 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.Be divided into after courage cell, CK-19 and albumin continue to express, and the expression of CK-7 is tending towards stopping (thinking a feature of more immature cell).On the other hand, liver cell maintains alpha-fetoprotein and albuminous expression, but does not express above-mentioned CK.Remain this example, the sign of stem cell is complicated as can be seen here, but preferentially service marking thing evaluates 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 breed in vitro and be divided in vivo liver cell (and preferably only having liver cell destiny) was not still described.In addition, former research is used complicated technology, as FACS, calcic medium or specific density gradient separate liver stem cells.

Therefore, a target of the present invention is just to provide 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 that separates described cell.

Summary of the invention

The invention provides progenitor cell or stem cell, the clone that comprises this progenitor cell or stem cell or the cell mass in the adult hepatic source obtaining from normal liver tissue.Before and after separating method, its culture, the differentiation of these cells, characterize, and 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 progenitor cell or stem cell (vertebrates of novel separation, preferred mammal, more preferably human cell), it is derived from adult hepatic, be characterised in that coexpression (to being positive below) liver cell mark albumin (ALB) and one or more of other livers or liver cell mark possibly, preferably CD29, alpha-fetoprotein (AFP), one in α-1 antitrypsin and/or MRP2 translocator, more than a kind of or whole marks, and at least one mesenchyme mark, especially mark CD90, CD44, CD73, one in vimentin (vimentin) and 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 one in the molecule of indication liver cell sample feature or function, more than a kind of or all: 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 one, more than a kind of or all marks: at least hematopoiesis mark CD45 and CD34 are negative, also can one or more other hematopoiesis marks, for example, CD105, HLA-DR are negative, bile duct cell epithelium mark Ck19 (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 one in the ovogonium core of one or two kernel, more than a kind of or all forms.

In one embodiment, the present invention provides the stem cell from adult hepatic of separation 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, the stem cell of separation 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 Expression of Albumin of antibody; 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 CD90 mark, have CD29 mark or have 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 stem cell that comes from the separation of adult hepatic presents CD90, CD73, CD29 and the CD44 positive and the albumin positive, the vimentin positive and α-smooth muscle actin positive.

The progenitor cell of separation or the method for stem cell or cell mass that the present invention also provides a kind of acquisition to comprise described progenitor cell or stem cell, the method comprises: adult hepatic or its part of (a) dissociating is to form primary cell group from described adult hepatic or its part, (b) by 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 of the liver stem cells or its cell mass that obtain separation, wherein comprise the following steps according to the inventive method: from adult hepatic culturing cell, therefrom isolating hepatocytes, bed board liver cell and cultivate 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 adult hepatic progenitor cell or the stem cell of the separation that uses 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, the clone of described separation is preserved in to 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 being provided by preservation people: ADHLSC).Therefore, the present invention relates to be preserved in cell, clone and the cell mass that BCCM preserving number is the separation of LMBP 6452CB (being called " LMBP 6452CB " clone herein), its subbreed comprises clone's subbreed, also relate to its offspring, comprising breaking up offspring, especially liver cell or liver cell like cell prepared therefrom, also relates to the derivative of its genetic modification.

The present invention also provides and comprises according to the present invention the hepatic progenitor cell that separates or the composition of stem cell or its cell mass.Preferably, liver cell is human hepatocytes, or mammiferous liver cell.

There are several important advantages according to progenitor cell of the present invention or stem cell (will say especially, 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 not controlled (tumour) that risk is lower while being used for the treatment of and increase or vicious transformation.

In addition, contriver finds, obviously do not show and (be for example divided into mesoblastema type according to progenitor cell of the present invention or stem cell, bone or chondrocyte, phoirocyte) ability, in the time using this cell or cell is implanted to liver organization, reduce this tissue dystopy form (ectopic formation).

The inventor also finds, 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 marker expression, 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 the gene therapy instructed of liver cell in useful.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 offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification) pharmaceutical composition and pharmaceutically acceptable carrier.Preferably, described liver cell is human liver cell, or mammalian liver cell.

The present invention is also 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 offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification).In one embodiment, hepatic diseases, includes but not limited to pku and other amino acid metabolism diseases (aminoacidopathies), hemophilia and other thrombin defects, familial hypercholesterolemia and other lipid metabolism disorders, urea cycle disorder, glycogenosis (glycogenosis), galactosemia, fructosemia (fructosemia), tyrosinemia (tyrosinemia), protein and carbohydrate metabolism defect, organic aciduria, mitochondrial disease, peroxysome and lysosome are not normal, protein synthesis is abnormal, liver cell translocator defect, glycosylation defect, hepatitis, liver cirrhosis, congenitalDy olism, acute hepatic failure, Acute Hepatic infects, Acute Chemical toxicity, chronic liver failure, cholangitis, cholehepatocirrhosis, Alagille syndrome, alpha-1-amtitrypsin deficiency, autoimmune hepatitis, Biliary atresia, liver cancer, liver cystic lesion, fatty liver, galactosemia, gallbladdergallstonecholetithiasis, Gilbert syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, and other virus infection type hepatitis, porphyria, primary sclerosing cholangitis, Reye Cotard, sarcoidosis, tyrosinemia, I type glycogen storage disease (type I glycogen storage disease), or Wilson's disease (Wilson ' s disease).

The present invention is also provided for therapeutic gene and expresses wrong method, the method comprises: (i) the functional copy of a gene is introduced to the group who is used to provide (wherein comprising the offspring of differentiation, especially liver cell or liver cell like cell) conversion according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring; (ii) group who transforms is at least partly introduced to patient's liver, this patient is just needing the functional copy of this gene.As an alternative, the group who transforms can be introduced to inhuman mammiferous liver, to produce the animal model of a new hepatic pathology.

The present invention is also provided for therapeutic gene and expresses wrong composition, described composition comprises according to the present invention introduces the hepatic progenitor cell of the conversion that has gene function copy or stem cell, its clone or cell mass or its offspring (comprising breaking up offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification).

The present invention is also provided for therapeutic gene and expresses wrong pharmaceutical composition, described pharmaceutical composition comprises introducing according to the present invention has the hepatic progenitor cell of gene function copy or stem cell, its clone or cell mass or its offspring (wherein to comprise the offspring of differentiation, especially liver cell or liver cell like cell), and pharmaceutically acceptable carrier.

The present invention is also 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 offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification).

The present invention also provides the liver supplementary unit that comprises shell (housing), described shell holds according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification).

The present invention also provides the method for carrying out in vitro 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 offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification) Contact test reagent, and observe (if any words) effect of at least one test agent to liver cell population.Preferably, at least one effect, comprising the effect to cell viability, cell function or this two aspect.

The present invention also provides the method for carrying out in vitro 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 offspring, especially liver cell or liver cell like cell, the optionally offspring of genetic modification) Contact test reagent, and (ii) observe (if any words) at least one relates to the variation of test agent after the predetermined testing period.Preferably, at least one changes, comprising test agent in the variation aspect structure, concentration or this two.

The present invention also provides and infects to being used for the treatment of liver the method that effective reagent is tested, 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 offspring with target infectious agent, especially liver cell or liver cell like cell, the optionally offspring of genetic modification) in order to infected group to be provided; (ii) test agent of the group's contact predetermined amount that makes to be infected, and (iii) observe (if any words) contact is to being subject to infect group's effect.In one embodiment, infectious agent comprises a kind of microorganism.In another embodiment, infectious agent comprises one or more of virus, 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, virus infection agent comprises hepatitis virus.

The present invention also provides a kind of productive target method of protein, described method comprises: (i) functioning gene of coding target protein is introduced according to hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring and (wherein comprised the offspring of differentiation, especially liver cell or liver cell like cell), (ii) be applicable to transcribing, translate and condition that optionally posttranslational modification occurs under hatch described cell mass, and (iii) results target protein.Preferably, liver cell is human liver cell.In one embodiment, target protein comprises vaccine antigen.

The present invention also provide in vitro or body in the method studied of growth to liver and hepatocyte differentiation, described method comprises: in vitro or in body, make according to the condition of hepatic progenitor cell of the present invention or stem cell, its clone or cell mass or its offspring (comprising breaking up offspring) Contact Effect differentiation condition and observe at least one effect to cell mass.

The method that the present invention includes cell, its preparation, sign, cultivation and produce.

The present invention also comprises by the culture of these cells of cryopreservation and carrys out these cells of preservation.

The present invention also comprises the technology of Transplanted cells in animal and the mankind.

The present invention also comprises according to the purposes of the next medicine for the preparation of the above-mentioned disease for the treatment of of liver stem cells of the present invention.The present invention also comprises the purposes of liver stem cells according to the present invention in test kit or part test kit.

Accompanying drawing explanation

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, as prepared by embodiment 1, uses opticmicroscope (differing) to observe afterwards through the cultivation of 1 month.A, lower converging (confluence); B, higher converges.Magnification is 100 ×.

Fig. 2 A: present and come from the progenitor cell of people's adult hepatic or the immunofluorescence dyeing of stem cell (ADHLSC) according to the present invention, as prepared in embodiment 1, after within 1 month, cultivating, 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 mankind's hepatoblastoma (HepG2,4 roads) are compared, the RT-PCR gene expression profile in 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 are divided into liver cell sample pedigree in vitro.Cell breaks up as described in Example 1, obtains image at the 2nd day (J2) of process, the 14th day (J14) and the 30th day (J30).

Fig. 4 represents, with several phenodins in the chimeric liver of the uPA/SCID mouse of undifferentiated ADHLSC Transplanted cells and eosin colored graph picture, as described in detail in embodiment 1, to show that these cells become the liver cell of differentiation.

Fig. 5 represents the image of several human albumins dyeing, shows in embodiment 1, transplants the liver cell sample group's who observes after 10 weeks humanized with undifferentiated ADHLSC.

Detailed Description Of The Invention

As used herein, unless the context clearly indicates otherwise, otherwise do not have the object meaning of quantifier restriction to comprise one and multiple referent.As an example, " cell " refers to one or more cell.

As used herein, term " comprises ", " comprising " and " containing " be synonym, is that have a pardon or open, and does not get rid of the extra member, key element or the method steps that do not describe in detail.

All reference of quoting in this specification sheets are all incorporated to herein by reference.The instruction of all references that especially relate to specially is herein by reference to being incorporated to.

obtain cell of the present invention

On the one hand, the invention provides a kind of method of the cell mass that obtains progenitor cell or the stem cell of separation or comprise described progenitor cell or stem cell, described method comprises: (a) dissociate adult hepatic or its part, to form primary cell group from described adult hepatic or its part, (b) by 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.

As used herein, term " cell of separation " typically refers to the cell of not being combined with one or more cells or one or more cellular constituent, and they are combinations in vivo.For example, the cell of separation can leave its natural surroundings, or can be from the propagation of cell of leaving natural surroundings, for example, and vitro proliferation.

As used herein, term " external " refers to outside animal or human's body or outside.Term used herein " external " is understood to include " in vitro ".Term " in vitro " typically refers to and leaves the tissue of animal or human's body or cell and preserve in vitro or propagation, as in culture vessel.

Term " cell mass " typically refers to one group of cell.Unless otherwise, otherwise this term refers to the cell colony that is made up of or comprises herein the cell separating herein the cell separating.

Cell mass can form maybe can comprise and has at least partly isophenic cell altogether by having altogether isophenic cell.When cell substantially similar or when consistent in one or more notable features, think that cell has common phenotype, its feature include but not limited to form outward appearance, certain cellular constituent or product (as RNA, protein or other materials) expression have or not or activity, multiplication capacity and/or kinetics, differentiation potential and/or the response to differentiation signal of level, certain biochemical route or vitro culture behavior (as, adhere to, non-cohesive, monolayer growth, growth kinetics etc.).Therefore this notable feature can define a cell mass or its part.

When claiming that cell mass is that " heterogeneity " is (heterogeneous) time herein, this ordinary representation cell mass comprises two or more and does not have isophenic cell or cell part altogether, the cell that comprises two or more different cell types as cell mass.Such as but not limited to, heterogeneity cell mass can separate from liver, and can comprise multiple liver cell type, include but not limited to liver cell (as large and little liver cell), bile duct cell, Kupffer cell, hepatic stellate cell (Ito cell) and liver endotheliocyte.

When claiming that cell mass is " homogeneous " (homogeneous) time herein, it forms by having isophenic cell altogether.The most cells that comprise while claiming cell mass " basic homogeneous " herein have common phenotype." basic homogeneous " cell mass can comprise at least 70%, as at least 80%, and preferably at least 90%, as at least 95% or even at least 99% cell has common phenotype, as the phenotype of specifically mentioning (as the phenotype of progenitor cell or stem cell).As used herein, therefore term used herein " basic homogeneous " also can comprise the group of homogeneous.

Term " cell mass that comprises progenitor cell or stem cell " relates to and comprises as defined here at least one progenitor cell or stem cell, in typical case the cell mass of part progenitor cell or stem cell as defined here.Conventionally, the progenitor cell of described part or stem cell can have common phenotype.

Term " progenitor cell " typically refers to does not have cell specialization or relatively less specialization and that have proliferation potential, and this cell or its offspring can produce at least one cell type of specialization relatively more.Such as but not limited to, progenitor cell can produce along the offspring of one or more pedigree differentiation and produce the relatively more and more cell of specialization, wherein this offspring and/or the relative more and more cell of specialization can self be exactly progenitor cell, or the cell that even the whole end of generation breaks up, that is: the cell of complete specialization, after this can be mitotic division.Term also comprises the stem cell of definition herein.

In the time thinking the another kind of cell of specialization relatively more of progenitor cell " generation ", such as but not limited to, progenitor cell is first through cell fission and be divided into another kind of cell, or another kind of cell through progenitor cell its offspring one takes turns or more wheels cell fission and/or differentiation after and produce.

The progenitor cell that term " stem cell " refers to can self (be undifferentiated and can breed), wherein the offspring of stem cell or at least its part substantially kept parental generation stem cell specialization or relatively less specialization phenotype, differentiation potential and multiplication capacity.This term comprises the stem cell of unlimited self substantially, that is: compare with parental cell, offspring or its part further ability of propagation significantly do not reduce, and the stem cell that shows limited self, that is: compare with parent cell, offspring or its part further ability of propagation significantly reduce.

Those skilled in the art will know that above-mentioned feature typically refers to progenitor cell and stem cell behavior in vivo, repeats out under suitable condition completely or at least in part in vitro and/or in vitro.

Based on the ability that produces different cell types, it is (unipotent) of all-round (totipotent), multipotency (pluripotent), specially energy (multipotent) or monoenergetic that progenitor cell or stem cell can be described to conventionally.Single " all-round " cell is defined as can grow (that is: growing) and becomes whole organism." multipotency " cell can not grow up to whole organism, but can produce the cell type that comes from all three germinal layers, in, in and ectoderm.And can produce all cells type of organism." specially energy " cell can produce from two or more Different Organs of organism or at least one cell type of tissue, wherein said cell type can be derived from identical or different germinal layer (germlayer), but can not produce all cells type of organism." monoenergetic " cell can only be divided into the cell of a clone.

Term " differentiation " or its derivative are for referring to a kind of process herein, and cell specialization or relatively less specialization becomes specialization relatively more by this process.In cell individual occurs, adjective " differentiation " is a relative terms.Therefore, " cell of differentiation " is the cell that a kind of growth enters specific development pathway than the cell of comparing.The cell of differentiation, for example, can be the cell that end breaks up eventually, that is: in the various tissues of organism and organ, bring into play the cell of the complete specialization of specialization function, but need not be after mitotic division.In another example, the cell of differentiation can be also the progenitor cell in differentiation pedigree, and it can further be bred and/or break up.Similar, if cell has entered specific development pathway than the cell development of comparing, cell is " specialization relatively more ", wherein the latter to be construed to be " not specialization " or " relatively less specialization ".The cell of specialization can be different from cell specialization or relatively less specialization aspect one or more remarkable phenotypic characteristics more relatively, described phenotypic characteristic as, for example, having or not or activity, form outward appearance, multiplication capacity and/or kinetics, differentiation potential and/or the response to differentiation signal etc. of level, certain biochemical route of the expression of certain cellular constituent or product (as RNA, protein or other materials), wherein these features show that the cell of specialization relatively is more further along the process of described development pathway.

The limiting examples of differentiation can comprise, as, myeloid-lymphoid stem cell become specified type special can progenitor cell or the variation of stem cell, specially can progenitor cell or stem cell become the monoenergetic progenitor cell of specified type or the variation of stem cell or monoenergetic progenitor cell or stem cell and become the cell type of specialization or the eventually variation of the cell of last specialization more in designated cell system.Can distinguish cytodifferentiation specialization or less specialization and the cell of specialization more by thering is the outward appearance of medium specialization degree cell.

Liver organization dissociates

As described in, method of the present invention comprises that dissociate adult hepatic or its part are to form primary cell group's step from described adult hepatic or its part.

Term " liver " refers to liver organ.Term " liver part " typically refers to any part of liver organ, for the region of the part of described liver organ or liver origin in amount without any restriction.Preferably, all cells type existing in liver organ also can occur in described liver part.The amount of part liver is deferred to actual Consideration on can be at least partly, as obtained the needs that are enough to the primary liver cell of implementing the inventive method.This consideration of instruction according to the present invention it will be apparent to those skilled in the art that.Therefore, such as but not limited to, part liver can represent (typically w/w) at least liver 0.1% or liver organ at least 1% or at least 10% or at least 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% or at least 90% or more than.In other limiting examples, part liver can be at least 1g or at least 10g at least 100g or at least 200g at least 300g or at least 400g at least 500g or at least 600g at least 700g or at least 800g at least 900g or at least 1000g at least 1100g or at least 1200g at least 1300g or at least 1400g or more than.For example, part liver can be lobe of the liver, for example, lobus dexter or lobus sinister, or liver split from operation in excision IV part.

Term used herein " adult hepatic " refers to and has obtained basic fully-developed tissue (tissueorganization) and cell composition.

Specifically, the known liver of those skilled in the art is experience growth variation in for some time after birth, and during this, liver obtains the tissue being mature on the whole.For example, in human subjects, connatae liver contains a considerable amount of hematopoietic cells, and it substantially disappears in 1-2 week after being approximately born from liver.And people's liver contains a group hepatic progenitor cell at birth, it is substantially replaced by mature hepatocytes and courage cell in some months after birth.

Therefore, in human subjects, " adult hepatic " referred in the rear any time of birth, the preferred full-time phase (full term), can be birth after at least one monthly age, as at least 2 months, at least 3 months, as at least 4 months, at least 5 months, as being at least born rear 6 monthly ages, as, for example 1 year or above, 5 years or above, at least 10 years or above, 15 years or above, 20 years or above or be born latter 25 years or the liver of above object.Therefore, can in human subjects, find " adult hepatic " or ripe liver, wherein human subjects will be described according to conventional term " baby ", " children ", " youth ", " teenager " or " adult " under different situations.

One skilled in the art will appreciate that liver can substantially reach maturity in different time in postpartum in different animals species, can proper interpretation term " adult hepatic " for each species.

Liver or its part obtain from " object ", " donor object " or " donor ", and wherein " object ", " donor object " or " donor " can exchange while relating to vertebrates, and preferred mammal, is more preferably people.

Term " Mammals " 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 animals, companion animals and laboratory animal, wherein laboratory animal as, for example, mouse, rat, rabbit, dog, cat, ox, horse, pig and primate, for example monkey and man like ape.

In an especially preferred embodiment, adult hepatic or its part are from human subjects.As described in detail in this specification sheets, can be advantageously used in from the progenitor cell of the liver of human subjects or stem cell or clone or its offspring according to the present invention, as, especially suffer from research and the treatment of the human patients of hepatopathy.

In another embodiment, adult hepatic or its part can be from inhuman animal targets, preferably non-human mammal object.Can be advantageously used in from the progenitor cell of non-human animal or non-human mammal object or stem cell or clone or its offspring according to the present invention, as, with it identical, relevant or other non-human animal or the species member of non-human mammal object in research and treatment hepatopathy, or even treatment suffers from the human patients of hepatopathy (as xenotransplant, the bioartificial liver devices that comprises non-human animal or non-human mammal cell).Such as but not limited to, the non-human mammal cell that is especially suitable for human treatment can be from pig.

Donor object can be live can be also dead, the principle of accepting according to this area is determined, as, for example, " heart-lung " standard (conventionally comprising circulation and the irreversible of respiratory function stops) or " brain death " standard (comprise full brain conventionally, 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 mankind's donor of living can need to meet and maintain donor survival from now on.Therefore, can only have in typical case a part for liver to separate from mankind's donor of living, as used examination of living tissue or excision, thereby in donor, maintain enough physiology liver function levels.On the other hand, obtain liver or its part possibility from non-human animal, but do not need to meet non-human animal's survival from now on.For example, after obtaining tissue, non-human animal just can have been put to death by humanity.These and similarly consideration be apparent for those skilled in the art, it has reflected law and moral standards and has not substantially related to essence of the present invention.

In one embodiment, liver or its part can obtain from donor especially mankind's donor, and it is as dirty in pulsatile heart that described mankind's donor has lasting circulation, and lasting respiratory function is as lung or the Spirophore breathed.Consider morals and Legal Norm, donor can need or not need brain death (as this may be unsuitable for the survival from now on of mankind's donor, but can allow to separate whole liver or its part in the mankind of brain death).From this donor, obtain liver or its part has superiority, do not suffer substantive anoxic (lacking oxygen) because organize, substantive anoxic is normally caused by ischemic (ischemia) (circulation stops).

In another embodiment, discovery as pleasantly surprised in the inventor, liver or its part can obtain from donor especially mankind's donor, described mankind's donor circulates and has just stopped in obtaining tissue, as heart stops beating and/or respiratory function stops, as the lung of not breathing and the machine that breathes no more.May suffer anoxic to a certain degree although come from liver or its part of these donors, the inventor find according to the present invention can with progenitor cell or stem cell also can obtain from these tissues.Liver or its part stop obtaining within latter about 24 hours in donor circulation (as heartbeat), as within about 20 hours, as within about 16 hours, more preferably within about 12 hours, within about 8 hours, even more preferably within 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, as donor circulation (as heartbeat) stops within latter about 45,30 or 15 minutes.

The tissue obtaining as above-mentioned can be cooled to about room temperature, or lower than the temperature of room temperature, but conventionally avoid freezing tissue or its part, especially this freezing meeting to cause nucleus to form or ice-crystal growth.For example, can at any temperature between about 1 ℃ and room temperature, between about 2 ℃ and room temperature, between about 3 ℃ and room temperature or between about 4 ℃ and room temperature, preserve tissue, and advantageously be kept at about 4 ℃.As known in the art, organize and also can be kept at " on ice ".Can be within ischemic stage all or in part (that is: stop for body-internal-circulation after time) cools tissue.In other words, tissue can be through the combination of the ischemic that is heated, cold ischemic or hot cold ischemic.Can before processing, the tissue obtaining be preserved and reach 48 hours, preferably be less than 24 hours, as be less than 16 hours, more preferably less than 12 hours, as be less than 10 hours, be less than 6 hours, be less than 3 hours, be less than 2 hours or be less than 1 hour.

Before further processing tissue, the tissue obtaining can be easily, but be not must be stored in as whole or be dipped at least partly suitable matrix and/or can but be not to pour into by suitable matrix.Those skilled in the art can select suitable can be before processing the matrix of sustentacular tissue's cell survival.

Method of the present invention comprises that the adult liver organization that dissociates as above forms 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 contact between cell and the cellular constituent of disorganize or organ.The object that it will be appreciated by those skilled in the art that disintegrated tissue or organ is from described tissue or Organ procurement cell (cell mass) suspension.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 obtain primary cell group (suspension) from it that is applicable to dissociating can be any method well known in the art, includes but not limited to enzymic digestion, mechanical separation, filtration, centrifugal and combination.In one embodiment, therefore the dissociate method of liver or its part can comprise that enzymic digestion liver organization discharges liver cell.In one embodiment, the dissociate method of liver or its part can comprise the physical disturbance to hepatic tissue or separate and discharges liver cell.In one embodiment, dissociate enzymic digestion that the method for liver or its part can comprise hepatic tissue and physical disturbance or both combinations of separating discharge liver cell.

The method of above-mentioned dissociate liver or its part is on the books in the art.For example, the method that separates liver cell from liver organization from nineteen sixty for mid-term be just known technology 1967.J Cell Biol 35:675-84 such as () Howard.Utilize the machinery of combination to separate rat hepatocytes with enzymic digestion technology, improve (J Cell Biol 43:506-20,1969) through Berry and Friend afterwards.This technology further develops and becomes two step collagenase perfusion (two-stepcollagenase perfusion) technology (Methods Cell Biol 13:29-83,1976) of widespread use through Seglen.

Therefore, in one embodiment, the method that dissociate liver or its part obtain primary cell group (suspension) from it is or comprises two step collagenase perfusion technology.Those skilled in the art will know that because described technology is above 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 in to existing main liver vessel, and fix by stitching.For part or the sections of liver, intubate can be placed in patient vessel's opening of cut surface, and fixes by stitching.In this case, conventionally need to seal little openings in blood vessels and prevent that primer solution is from cut surface seepage.With the free damping fluid perfusion of the divalent cation liver organization of preheating at 37 ℃, 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 as sodium-chlor and Repone K etc.This causes cell to maintain desmosome structural damage together.Then use damping fluid perfused tissue, damping fluid contains divalent cation, as Ca ²⁺and Mg ²⁺, and the extracellular matrix degrading enzyme of performance digestion function of organization.Primary liver cell, especially liver cell is conventionally released mechanicalness through gentle physical disturbance and completes dissociation process, as with comb rake, vibration, with 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, about 0.1 millimeter or above, approximately 0.25 millimeter or above, approximately 0.50 millimeter or above, approximately 1 millimeter or above, approximately 2,3,4 or 5 millimeters.A series of strainers that can reduce gradually with mesh make tissue dissociate and release cells step by step.The cell dissociating with damping fluid rinsing, damping fluid contains proteinase inhibitor, serum and/or blood plasma and comes deactivation collagenase and other filling process enzyme used, separate through low-speed centrifugal, as between 10 × g and 500 × g, (all viable cell can precipitate easily substantially, and dead cell and cell debris are removed substantially), wash with ice-cold damping fluid the throw out obtaining and carry out purifying cells suspension.

The hepatocellular quality and quantity separating depends on as the difference of the quality of tissue used, the perfusion composition of damping fluid and the type of enzyme and concentration and difference.Conventional enzyme comprises, but be 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 conventionally, can, conventionally by the compositions of mixtures of the enzyme without fine purifying, wherein can have inconsistent enzyme effect.Some enzyme shows protease activity, may cause undesirable reaction and affect quality and the quantity of vigor/healthy cell.Those skilled in the art will know that and obtain alive liver cell group with the enzyme of enough purity and quality.

The additive method that obtains primary liver cell can not comprise enzymic digestion technology.Mechanicalness is broken and is used widely, although the output of the liver cell of producing by this process is often less than and is obtained by collagenase digesting, and less consistence.But nearest method has been developed and had remarkable achievement, 2002.Cell Biol lnt 26:1003-1006 such as () Kravchenko comprising in cooling environment, combines the perfusion of sucrose-EDTA with controlled vibration.With the sucrose solution situ perfusion liver that contains EDTA (pH7.4).After perfusion, liver is separated from health, be placed in dish, segmentation packs in the ice-cold matrix of small volume.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 with coarse net filtering the initial suspension of liver cell subsequently.Cell can be suspended in 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 at least discharging liver cell from liver organization.The cell suspension obtaining 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 for obtaining 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 can have at most about 100% independent cell, unicellular.

As mentioned, thus the liver organization that dissociates provides primary cell group from described adult hepatic or its part.

As use herein, term " primary cell " comprise from object tissue or organ as the cell of existence in the cell existing the cell suspension that (be bed board before cell mass) obtains by dissociating, outer planting tissue, when the bed board first before two types cell, from the cell of the cell suspension of the cell of bed board first.Term " subculture cell " refers to the cell in all subsequent steps in cultivation.Therefore,, when the primary cell of bed board goes down to posterity first, as from substrate surface picking bed board again, as the follow-up middle all cells that goes down to posterity, be referred to herein as subculture cell so.

Primary cell group definition herein and that obtain by dissociate liver or its part is normally inhomogenous, can comprise the cell that belongs to cell type in more than one livers that is:.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 illustrating, " 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 author proposes, the liver cell of " greatly " of its definition is the parenchyma of being responsible for liver physiology function, and the liver cell of " little " provides the storehouse that a growth is hepatocellular progenitor cell (to see, as BiochemBiophys Res Commun 214:310-7 such as Mitaka, 1995).Also still unrestricted as further illustrating, " bile duct cell " comprises the epithelial cell of bile duct.Also still unrestricted as further illustrating, " elliptocyte " comprise unique form (as, karyomorphism) and cell sign thing express cell, as well-known in this area institute, being considered to is the progenitor cell that can produce liver cell and bile duct cell (the .KN.2003.J Gastroenterol Hepatol 18:4-12 such as Lowes under given conditions; The 1999.J of Hepatology 31:497-507 such as Yi).

Therefore, in one embodiment, inhomogenous primary 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 the cell of all or substantially all liver-composing type cell types, include but not limited to cell type listed above.Those skilled in the art understand the liver cell type that inhomogenous group describes before can comprising, no matter in body or external, and this area liver cell type of not yet describing in the past, and classified, separate and/or characterizing.

Those skilled in the art also know that 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 substantially same as the relative proportion existing in its liver that separates place or its part.For example, 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, the cell suspension wherein obtaining can be carelessly or targetedly enrichment aforesaid one or more cell types.In addition, some dissociating method can have different impacts to the survival of different liver cell types and/or vigor.Technician also know for one or more target liver cell types and for enrichment by the method for this area of the cell mass that separates liver or its part and obtain.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, the uneven a group of primary liver cell can comprise the cell that belongs to all or substantially all liver-composing type cell types.The inventor finds the method for undocumented hepatic progenitor cell before obtaining or stem cell type.In any case contriver does not wish that the source of described novel progenitor cell or stem cell is subject to the restriction of any hypothesis.

Such as but not limited to, described progenitor cell or stem cell or its ancestors are Already in liver, as liver essence or non-essence.For example, this ancestors can have and the progenitor cell separating or identical, the similar or different phenotype of stem cell (as cultivation according to the present invention can change ancestors' phenotype).As an alternative, or in addition, the progenitor cell of separation or stem cell can be because of for a change 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 the cell of all or substantially all liver cell types.

Contriver has been found that can be from obtaining progenitor cell of the present invention or stem cell by separating the cell mass that liver or its part form easily, and wherein said cell mass comprises liver cell.Therefore, the applicable method that separates liver or its part according to the present invention forms and comprises hepatocellular cell mass.Be not bound by any theory, contriver thinks that hepatocellular mode together discharges from liver organization (liver obtains by separating) at least to discharge from liver for the progenitor cell of invention or stem cell or its ancestors.

In one embodiment, the method of liver or its part of dissociating can form and comprise the hepatocellular cell mass of certain proportion, wherein at least about 10%, at least about 20%, at least 30%, at least 40%, preferably at least about 50%, as at least 60%, more preferably at least about 70%, as at least about 80%, even more preferably at least about 90% or above, as at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.The inventor find to form the liver cell that comprises significant proportion (as, as mentioned above at least about 50% or more than) separation liver or the method for its part provide applicable initiator cell group for obtaining 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 the further step for 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 for liver cell and/or other cell types especially liver cell and enrichment of cell group's step obtain and comprise the hepatocellular cell mass of aforementioned proportion.But, technician knows, in the method for 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 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 enrichment liver cell and/or especially liver cell of other cell types, thereby retains progenitor cell of the present invention or stem cell or its ancestors in the cell mass obtaining, and this belongs in those skilled in the art's 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 the cell mass obtaining from liver, be able to enrichment by suitable separation method.Based on one or more principles determine separate or for progenitor cell of the present invention or stem cell or its ancestors and within the cell mass fraction (fraction) of enrichment belongs to those skilled in the art's limit of power.This can cultivate the 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 refers to so a kind of cell mass, its neutralization can be in vivo or the cell of finding in the cell mass of enrichment compare, one or more cell types exist with larger relative proportion.

Bed board is from the primary cell of liver organization

Method of the present invention comprises cultivates the primary cell group who obtains by the method for the liver organization that dissociates as described here.For this reason, the primary cell group of liver cell is plated on and in substrate, makes cell attachment on it.

Term used herein " bed board " and inoculation synonym, and typically refer to by cell mass introduce can promote introduce cell survival and/or the external environment of growth.Conventionally, in the system that described environment can suitably distinguish in a kind of and surrounding environment, provide, thereby, can avoid the exchange of substance between described environment and 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 after cultivating harvested cell etc.) of continuous or intermittent other useful matter compositions between described environment and surrounding environment.Conventionally, can in culture vessel known in the art, set up the environment that is suitable for culturing cell, as, for example, various forms of cell cultures shaking flasks, orifice plate and plate.

In the present invention, cell (as primary liver cell) is plated on and allows the substrate of cell attachment on it, cell attachment or attaching are not repelled in its surface.This can by as cell is plated in culture systems (as culture vessel) and is implemented, wherein said culture systems presents one or more and is suitable for the substrate surface of cell attachment.In the time that the cell suspension (as the suspension in matrix) of culture systems is introduced in described one or more substrate surface contacts, can there is cell adhesion between cell and substrate surface.Therefore, term " is inoculated in cell to allow the substrate of cell attachment on it " and refers to cell is introduced to culture systems, the substrate surface that is characterized as at least one extensive compatible cell adhesion of described system, thereby the cell of inoculation can contact described substrate surface.The rule that maintains the cell culture of adhesion is being known in the art.

Conventionally, allowing the substrate of cell attachment on it can be any very hydrophilic substrate.As known in the art, culture vessel, as cultivate shaking flask, orifice plate, plate etc., conventionally can be formed by various macromolecular material manufactures, 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.Conventionally the culture vessel making with this material will carry out surface treatment in order to hydrophilic base surface to be provided after cast molding, thereby improves the possibility of effective cell attachment.Surface treatment can take top coat to process maybe can to relate on the surface use by directional energy to wish to produce at polymer surfaces the mode of chemical group.These chemical groups have general affinity to water, or show enough polarity and make its stable other polar group that is adsorbed to.These functional groups cause wetting ability, and or increase Surface Oxygen and think and can improve the character of cell in the substrate surface growth of this modification.This chemical group can comprise as amine, acid amides, carbonyl, carboxylic acid, ester, hydroxyl, sulfydryl etc.The example of directional energy comprises 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 at present attached cell comprises the chemical mediator that uses definite ingredients, and is added with ox, the mankind or other animal serums.Add serum except nutrient and/or somatomedin are provided, can also by be easier to one deck cell adhesion base coating (coating) process frosting promote cell adhesion.

The substrate surface of compatible cell adhesion as an alternative can be a glass, optionally, introduces the surface that strengthens its hydrophilic processing as functional group listed earlier.

Other adhere to substrate surface and can produce through top coat processing, 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, ECM protein fibre, ln, collagen protein (preferably I type albumen), glycosaminoglycan (as heparin or Suleparoid), fibronectin splicing variants (fibronectin), gelatin, vitronectin (Vitronectin), elastin, Gu raw albumen (Tenascin), can aggrecanase (aggrecan), agrin (agrin), Bone sialoprotein (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 element or its multiple combination.

Preferred embodiment can comprise scleroproein, ln or collagen protein.Other preferred embodiments can relate to the component that comprises ECM composition, as Matrigel basement membrane matrix (BDBiosciences), it is the solubility basilar membrane prepared product extracting from EHS murine sarcoma, described EHS murine sarcoma is rich in ECM protein, and wherein main component is ln, is secondly IV Collagen Type VI, heparan sulfate proteoglycan and nidogen.

Particularly preferred embodiment comprises the coating 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 the density inoculating cell with an expectation subsequently, counting cells possibly.Wherein, in the present invention, after inoculation, cell can mainly adhere to (as culture vessel) on the substrate surface existing in culture systems, with every mm ²or cm ²the cell number of described substrate surface inoculation represents inoculum density.In the present invention, the inoculum density of the primary cell obtaining from liver or its part of separation 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 primary liver cell inoculation, make cell suspension contact adhesive surface to allow cell to adhere to described substrate from cell mass.Primary liver cell with adhere in the contacting of substrate, can be conveniently by cell suspension in the environment that contains matrix at least, in method of the present invention, be fluid matrix in typical case, it contributes to survival and/or the growth of cell.Can be by matrix introducing before cell, simultaneously or add afterwards system.Matrix can be fresh, that is: previously not for culturing cell maybe can comprise at least a portion by cell cultures in advance (as, cultivate be about to bed board or existing cell before, or cultivate the cell not too relevant or irrelevant with the cell of inoculating) part of institute's adaptive processing (conditioned).

For the ease of described adhesion, in one embodiment, 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, primary cell suspension can contact with adhesive surface between approximately 2 hours and approximately 48 hours, according to appointment between 12 hours and approximately 48 hours, preferably between approximately 12 hours and approximately 36 hours, according to appointment between 16 hours and approximately 32 hours, even more preferably from about between 20 hours and approximately 28 hours, most preferably from about 24 hours.

Although the preferred above-mentioned time, also can provide shorter or longer time for being suitable for the adhesion of cell of the present invention, and those skilled in the art can optimize this time.

Make from primary liver cell group's cell adhesion, to above-mentioned adhesion substrate, NA material to be 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 the cell of adhesion, or within the time allowing adherent cell not), do not have great-hearted or dying cell, cell debris etc.Can remove NA material by discard matrix from system in typical case.The cell adhering to still sticks in substrate, optionally uses suitable matrix or isotonic buffer solution (as PBS) rinsing once or repeats rinsing attached cell and culture systems.At this, select to adhere to substrate surface, from primary liver cell group's cell for further cultivating.

Bed board cell and allow the environment of cell adhesion can comprise at least one matrix, fluid matrix normally in method of the present invention, its sustenticular cell survival and/or growth.Can be by matrix introducing before cell, simultaneously or add afterwards system.Matrix can be fresh, that is: previously not for culturing cell maybe can comprise at least a portion by cell cultures in advance (as, cultivate be about to bed board or existing cell before, or cultivate the cell not too relevant or irrelevant with the cell of inoculating) 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 with cultivate subsequently the step of attached cell in matrix used composition identical or basic identical.Or matrix can be different.Advantageously, matrix can comprise serum or blood plasma, and this can further promote cell adhesion.

Cultivate the primary cell from liver organization

Adhere to the cell from 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 maintaining and/or growing of cell and/or its offspring.

In some embodiments, can cultivation of primary cells between at least about 10 days and approximately 40 days, preferably at least about between 15 days and approximately 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 extending can need regularly substratum to be replaced by fresh culture in culture systems.Those skilled in the art can judge whether to change substratum by checking cell cultures parameter (as the outward appearance of pH value, cell density or cell).In typical case, conventionally can regularly replace matrix, as, every 1 to 10 day, preferably postvaccinal 16 to 32 hours (as, approximately 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 optionally, thereby retain the part matrix of being adjusted by previous cell cultures.In one embodiment, the matrix of substantially all volumes is all replaced by fresh matrix.In a further preferred embodiment, during the cell cultures extending, do not change matrix.

There is cultivation of primary cells suspension and further adherent cell under liquid nutrient medium condition.In typical case, matrix comprises basic medium formula well known in the art.Many basic medium formulas (can be from as American Type Culture Collection, ATCC, or Invitrogen company, Carlsbad, 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), BGJb substratum, F-12 Nutrient 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 adjust matrix and/or fill-in concentration for cultured cells, also within those skilled in the art's limit of power.In one embodiment, preferably basic medium formula can be Williams Medium E, and it is a kind of nutritious formula, is reported in externally can maintain adult liver cell culture.Other embodiments can adopt other basic medium formulas, as are selected from above-mentioned.

This basic medium formula 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, as sodium-acetate) etc.Obviously, much available matrix be contain or containing Sodium.alpha.-ketopropionate low-glucose formula.

For cultivating, basic medium can be added with a kind of and multiple other compositions.For example, extra supplementing can provide necessary trace element and the material for the most suitable growth and amplification to cell.This supplementing comprises Regular Insulin, Transferrins,iron complexes, selenium salt and combination thereof.These compositions can be included in 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 supplement 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, Liu Suanyan NEOMYCIN SULPHATE, 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 supplementing 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 etc.Albumin can be similarly for the formula of FAF.

It is also conceivable that and in cell culture medium, supplement mammiferous blood plasma or serum.Blood plasma or serum often contain vigor and the necessary cytokine of amplification and composition.Also it is suitable to serum to consider to use.

Term " blood plasma " is as defined in routine.Blood plasma normally obtains from whole blood sample, extracting when blood sample or in the near future, provide 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 " refers to the composition that does not form the mankind or an animal body part.

Term " serum " is as defined in routine.Serum normally obtains from whole blood sample, first allows sample generation blood coagulation, subsequently by suitable technology, by centrifugal, the cellular constituent of formed grumeleuse and blood sample is separated from liquid component (serum) in typical case.Inert catalyst, as granulated glass sphere or glass powder, can promote blood coagulation.Advantageously, can pass through serum separator well known in the art (serum-separating vessel; SST) prepare serum, the catalyzer that described SST contains inertia promotes blood coagulation, also comprises that density sets the glue between centrifugal rear liquid component and grumeleuse and the density of cellular constituent, thereby has simplified separation.As an alternative, can be by removing antithrombotics and scleroproein obtains serum from blood plasma.Therefore term " serum " refers to the composition that does not form the mankind or an animal body part.

The blood plasma or the serum that separate can be directly used in method of the present invention.In method of the present invention, blood plasma or the serum that also can properly preserve separation give over to rear use.Can preserve in the short period of time in typical case blood plasma or serum, as, reach about 1-2 week, higher than blood plasma or serum zero pour but preserve under room temperature lower than surrounding enviroment temperature separately.Conventionally, approximately 15 ℃ of this temperature or following, preferably approximately 10 ℃ or following, more preferably from about 5 ℃ or following, as, approximately 5 ℃, 4 ℃, 3 ℃, 2 ℃ or approximately 1 ℃, most preferably from about 5 ℃ or approximately 4 ℃.As an alternative, blood plasma or serum can be stored in lower than the temperature of zero pour, i.e. freezing preservation separately.As common in this area, can be approximately-70 ℃ or following for the favourable temperature of freezing preservation blood plasma or serum, as, approximately-75 ℃ or following or approximately-80 ℃ or following.This temperature can favourablely prevent preserve blood plasma or any of serum thaw, thereby keep its quality.No matter how long blood plasma or serum need to preserve period, can adopt freezing preservation.If but while needing longer-term to store, as exceed a couple of days or exceed 1-2 week, freezing preservation is especially applicable.

Before preserving or using, the blood plasma or the serum that can heat inactivation separate.Heat inactivation is mainly used in removing fill-in in the art.Hot deactivation generally includes, and allows blood plasma or serum be cooled to gradually after envrionment temperature, and blood plasma or serum are hatched 30 to 60 minutes at 56 ℃, as 30 minutes, and continues to mix.Those skilled in the art will know that any conventional the improvement and demand of said process.

Optionally, also can before preservation or use, blood plasma or serum be carried out to sterilizing.Common sterilizing means can comprise, as, the strainer that is less than 1 μ m by one or more apertures filters, preferably be less than 0.5 μ m, as be 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 less, as 0.15 μ m or less, 0.10 μ m or less.

Can comprise human serum or blood plasma for suitable serum or the blood plasma of matrix of the present invention; the serum of non--mankind's animal or blood plasma; preferably non--mankind's Mammals; for example; as, serum or the blood plasma of inhuman primate (as mongoose lemur, monkey, orangutan), tire ox or Adult Bovine, 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 with obtaining primary liver cell, obtain serum or blood plasma.In a limiting examples, human serum or blood plasma can be used for cultivating primary human liver cell.

In a further preferred embodiment, matrix comprises serum or blood plasma, preferably tire ox (calf) serum or blood plasma, more preferably tire ox (calf) serum (FCS or FBS).

In another embodiment, comprise bovine serum or blood plasma for cultivating the matrix of primary human liver cell, preferably tire ox (calf) serum or blood plasma, more preferably tire ox (calf) serum (FCS or FBS).

In one embodiment, matrix comprises serum between approximately 0.5% and approximately 40% (V/V) or blood plasma or for serum, preferably between approximately 5% and approximately 20% (V/V), as, between approximately 5% and approximately 15% (V/V), more preferably from about between 8% and approximately 12% (V/V), as, approximately 10% serum or blood plasma or for serum, especially preferred serum defined above or blood plasma.

In other preferred embodiments, comprise for the matrix of cultivating primary human liver cell, amount is between approximately 0.5% and approximately 40% (V/V), preferably between approximately 5% and approximately 20% (V/V), as, between approximately 5% and approximately 15% (V/V), more preferably from about between 8% and approximately 12% (V/V), as, approximately 10% bovine serum or blood plasma, preferably 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 cultivate corresponding species, the serum of another species or the mixture of blood plasma of primary liver cell.For example, can comprise the mixture of human plasma or serum for the matrix of the primary liver cell of cultivator, preferably human serum, and Ox blood plasma or serum, preferably bovine serum.

In addition, matrix can preferably comprise the somatomedin that at least one exogenous (except blood plasma or serum) adds.Technician knows the basic medium conventional ingredient of (before adding serum or 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 " refers to that a class affects various kinds of cell type propagation, growth, differentiation, existence and/or migration, can affect the biologically active substance that organism grows, morphology and function changes, its independent role or regulated by other materials.Somatomedin usually used as part the acceptor by the response somatomedin in conjunction with existing in 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 a 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-binding 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.In a particularly preferred embodiment, matrix comprises EGF.

In another embodiment, matrix comprises a kind of somatomedin, and it is the somatomedin that Regular Insulin is relevant (IGF) family member.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.In a particularly preferred embodiment, matrix comprises Regular Insulin.

In another embodiment, matrix comprises a kind of somatomedin, and it is 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.In a particularly preferred embodiment, matrix comprises dexamethasone.

In another preferred embodiment, the somatomedin that matrix comprises any two or more exogenous interpolations or the herein combination of the defined preferred growth factor.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 of the somatomedin that comprises these exogenous interpolations can preferably include the serum or the blood plasma that in above-mentioned embodiment, define.

Under what concentration, the particular growth factor can be induced the especially effect of the cell to vitro culture, and this concentration can be used for above-mentioned somatomedin, and this concentration is general knowledge to those skilled in the art.For example, rather than restriction, in typical case can be approximately between 0.1ng/ml and 1 μ g/ml, preferably between 1ng/ml and 100ng/ml, as, under about 25ng/ml concentration, use EGF; In typical case can be between about 0.1 μ g/ml and 1mg/ml, preferably between approximately 1 μ g/ml and 100 μ g/ml, as, under approximately 10 μ g/ml concentration, use Regular Insulin; Can, at about 0.1nM and 1 μ M, preferably, at about 1nM and 100nM, under 10nM concentration, use dexamethasone according to appointment in typical case.

In a preferred embodiment, especially in the method for human liver cell, the present invention's somatomedin used can be human growth factor.Term " human growth factor " refers to a kind of and the essentially identical somatomedin of naturally occurring human growth factor as used herein.For example, in the time that somatomedin is protein entity, its composing type peptide or polypeptide can have the amino acid primary sequence consistent with naturally occurring human growth factor.Preferably use the human growth factor of the inventive method, estimate that this somatomedin causes the effect of the cellular function of expecting.

As described in, the inventor has been found that by continuing cultivation one period of primary liver cell (period as defined above), preferably use above-mentioned matrix composition, occur progenitor cell of the present invention or stem cell propagation, and the advantage of the liver cell type of differentiation in the primary liver cell extending is cultivated reduces.Not limited by any hypothesis, the primary liver cell type of differentiation is passable, for example, when Extending culture, could not breed, dead and/or contrary differentiation (retro-differentiation).As implementation section describes in detail, can pass through, wherein, other cell types that exist in differentiating forms progenitor cell or stem cell and primary cell culture, according to the inventor's knowledge, described Shape Representation is mesenchyme or mesenchyme sample form, in typical case, comprises flat pattern, obvious tenuigenin and/or have the ovogonium core of one or two kernel.

The inventor also finds further to promote by changing culture medium appearance, propagation and the enrichment of described progenitor cell or the former culture of stem cell, thereby be conducive to further eliminate the liver cell type of one or more differentiation, especially liver cell, liver cell can occupy leading in the primary liver cell culture separating.In this case, progenitor cell of the present invention or stem cell can advantageously breed and become the predominant cell types in primary liver cell cultivation.As, weaken 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, due to the contrary differentiation in the training period of one or more cell types, the phenotype of differentiation can be weakened.

In the time maintaining the propagation of progenitor cell of the present invention or stem cell (for example, can easily judge for the visual inspection cell cultures of passing through of different cell types), can use any especially hepatocellular matrix of liver cell type that is conducive to eliminate 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 concentration, if concentration is between 3000mg/1 and 6000mg/l, and between preferred 4000mg/l and 5000mg/l, and about in typical case 4500mg/l.Another kind of variation can be the somatomedin that there is no exogenous interpolation (, except existing in serum or blood plasma).For example, in matrix, at least there is no Regular Insulin, dexamethasone and/or the EGF of exogenous interpolation.Another kind of variation can be to use the basic medium in addition except Williams Medium E (being especially applicable to long-term cultivation primary liver cell type, especially liver cell).For example, rather than restriction, as the use of the basic medium of MEM, DMEM, alpha-MEM or EMEM can have superiority.Need to be appreciated that, can change matrix by above-mentioned a kind of, more than one or all modes.Also need to be appreciated that, matrix comprises above-mentioned serum or blood plasma or for serum, comprising the preferred embodiment describing in detail above.

In one embodiment, can add at the beginning the matrix that is conducive to eliminate the liver cell type of above-mentioned one or more differentiation and promotes progenitor cell of the present invention or stem cell at the primary liver cell of cultivation.In another embodiment, can during the primary liver cell of Extending culture, change in this way matrix.For example, can be in inoculation approximately 1 day, approximately 2 days, 3 days after primary liver cell, as, approximately 4 or 5 days, or originate in approximately 6 days, for example, as, approximately 7 or 8 days, or originate in approximately 9 days, for example, as, approximately 10 or 11 days, originate in approximately 12 days, as, approximately 13 or 14 days, more preferably originate in approximately 15 days, as, approximately 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, mode changes after matrix according to this, can be as, and every 2 to 6 days, preferably every 2 to 4 days, as, changed/upgrade matrix every 3 or 4 days.

Therefore, in an exemplary preferred embodiment, after bed board, can in the matrix that contributes to primary liver cell survival, cultivate primary liver cell, described primary liver cell comprises the liver cell type of differentiation, as liver cell, in the time of the above-mentioned time, matrix can be changed into and contribute 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, primary liver cell can first be incubated to be had in a kind of, matrix more than a kind of or all following features: comprise a kind of nutritious basic medium, for example, as WilliamsMedium E, comprise low concentration glucose, as between 500mg/l and 2999mg/l, preferably in the middle of 1000mg/l and 2000mg/l, comprise the somatomedin of at least one exogenous interpolation, preferably the one in Regular Insulin, dexamethasone and EGF, 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 Williams Medium E, for example, as the basic medium of MEM, DMEM, alpha-MEM or EMEM, comprise height (concentration) glucose, not containing the somatomedin of exogenous interpolation or at least not containing dexamethasone, Regular Insulin and/or EGF.Need to be appreciated that, above-mentioned matrix will comprise above-mentioned serum or blood plasma or for serum, comprising the preferred embodiment describing in detail above.

As described in, the cultivation of above-mentioned primary liver cell causes generation and the propagation of in culture progenitor cell of the present invention or stem cell.Described cultivation can be maintained easily until the progenitor cell of the present invention occurring or stem cell fully breed.For example, can continue described cultivation until cell mass arrives certain degree of converging, 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 refers to culturing cell, and the cell contacting with each other under this density covers all surfaces (, converging completely) for growth substantially.

Passage

After the generation and propagation of above-mentioned primary liver cell cultivation and progenitor cell of the present invention or stem cell, the cell mass so obtaining can at least go down to posterity once.

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 primary cell culture, thereby allows the cell mass of further these cells of enrichment easily.For the purpose of convenient, what this one-phase of described method carried out go down to posterity refers to " going down to posterity first " (the going down to posterity 1) in the inventive method herein.Cell can go down to posterity at least one times, 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 and represents, as goes down to posterity 2,3,4,5 etc.

While going down to posterity, culturing cell dissociates each other (detach) and separates from culture medium.Can carry out dissociating and separating of cell according to (method) well known in the art, (as be selected from trypsinase, collagenase as carried out enzyme processing with proteolytic enzyme, as I, II, III or IV type, Dispase, pronase (pronase), papoid etc.), with divalent ion sequestrant (as, EDTA or EGTA) or mechanicalness processing (as, by small-bore pipettor or move liquid head and repeatedly blow and beat), or the arbitrary combination of these processing is processed.Preferably, dissociating of culturing cell will produce the individual cells of significant proportion with separating.For example, 40% or the above cell reclaiming 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 relatively less cell, as, on average more than the cell between 1 to 10, as be less than 8 cells, preferably be less than 6 cells, more preferably less than 4 cells, as be less than 3 or be less than 2 cells.

In general, a kind of method that is suitable for Redispersion cell should retain cell viability.Preferably, the cell suspension obtaining after Redispersion can comprise at least 60% viable cell, as, at least 70%, more preferably at least 80%, most preferably at least 90% to 100% viable cell.Those skilled in the art will know which kind of condition of common selection guarantees cell dissociation and the dispersion of expected degree, preserve cell viability.

Afterwards, by the cell dissociating and separate (in typical case, suspension in isotonic buffer or matrix) renewed vaccination to matrix, make cell adhesion thereon, be incubated at subsequently as mentioned above and in matrix, maintain progenitor cell of the present invention 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, preferably 1 × 10 ³with 1 × 10 ⁵individual cell/cm ²between, as, approximately 1 × 10 ³individual cell/cm ², approximately 5 × 10 ³individual cell/cm ², approximately 1 × 10 ⁴individual cell/cm ², approximately 5 × 10 ⁴individual cell/cm ², or approximately 1 × 10 ⁵individual cell/cm ², preferred approximately 1 × 10 ³with 1 × 10 ⁴individual cell/mm ²between inoculum density renewed vaccination cell.

As an alternative, between approximately 1/8 and 1/2, between preferred approximately 1/4 and 1/2, more preferably from about 1/2 or approximately 1/3 ratio of division (splitting ratio) renewed vaccination cell.Ratio of division represents the ratio of the cell going down to posterity, and is seeded 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 described in detail in this explanation.Matrix is identical with the matrix of the primary liver cell of bed board (comprising the preferred embodiment of above-mentioned this matrix) or difference preferably.Preferred this matrix is collagen protein, especially above-mentioned type i collagen albumen.

Further the cell of subculture is until cell becomes at least 50% converges, as, at least 60%, preferably at least 70%, example, at least 80%, more preferably at least 90%, as, have 95% or converge even completely at least.

The inventor has been found that progenitor cell of the present invention and the stem cell that the cell mass of this one-phase acquisition of described method comprises significant proportion, cell can advantageously go down to posterity once (that is: at least second pass generation) at least again, substantially as abovely goes down to posterity for the first time.By form and/or molecular marker judgement, 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 cultivate primary liver cell after, cause progenitor cell or generation and the propagation of stem cell in described culture, (cultured cells goes down to posterity at least one times, go down to posterity first) preferably at least twice (for the first time with second pass generation), optionally more times go down to posterity (for the first time, for the second time and follow-up go down to posterity) at every turn.For example, after inoculating primary liver cell, cell can go down to posterity at least one times, at least 2 times, at least 3 times, at least 4 times or at least 5 times.In another embodiment, after inoculating and cultivating primary 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.With go down to posterity for the first time, as above-mentioned, under basically identical or similar condition, carry out 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 apparent improvement to those skilled in the art.

Therefore method of the present invention provides the progenitor cell as defined in the disclosure that comprises significant proportion or the cell mass of stem cell, and the ratio of described progenitor cell or stem cell is along with the one or many of primary liver cell Extending culture goes down to posterity and increases.In typical case, described group will comprise at least about 10%, as, at least about progenitor cell described in 20% or stem cell, but contriver finds conventionally will obtain 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% or at least at least 90% or more than.In addition, method even can produce described progenitor cell or the population of stem cells of basic homogeneous or homogeneous.Can evaluate by any suitable standard method the ratio of progenitor cell or stem cell, as pass through flow cytometer.

Maintain the cell of acquisition

As the progenitor cell of the present invention that contains liver source by method acquisition of the present invention or the group of stem cell, and can so enrichment described in when progenitor cell or stem cell, can be next maintain and/or proliferating-cell population under undifferentiated condition allowing described progenitor cell or stem cell growth and multiplication.This condition can be, as, for obtaining the condition of progenitor cell or stem cell.The those skilled in the art whether cytodifferentiation occur can be passed judgment on and other conditions can be set up easily.This contributes to increase due to the available progenitor cell of subsequent use or the number of stem cell.

The inventor have been found that can frozen (as for mammalian cell, this is known) primary or any other follow-up going down to posterity for purposes subsequently.

The inventor has been found that the progenitor cell of the present invention or the stem cell basic multiplication capacity that keeps after freeze thawing that in primary cell culture, occur.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 description, be thawed and bed board (cell) again.

progenitor cell of the present invention or stem cell

The inventor find by cultivation described above and preferably also obtain by the primary liver cell that goes down to posterity the cell mass progenitor cell or the stem cell that comprise liver of the present invention source, at least one mesenchyme mark of its coexpression (to being positive below), especially for example CD90, CD73, CD44, one in vimentin and α-smooth muscle actin (ASMA), more than one, as 2, 3, or 4 kinds or whole, and liver cell mark albumin (ALB) and one or more of other livers or liver cell mark possibly, preferably CD29, alpha-fetoprotein (AFP), one in α-1 antitrypsin and/or 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 one mesenchyme marks of coexpression (to being positive below), especially CD90, CD73, CD44, one in vimentin and α-smooth muscle actin (ASMA), more than one, as 2, 3, or 4 kinds or whole, and liver cell mark albumin (ALB) and one or more other liver cell marks possibly, preferably one or both in α-1 antitrypsin and MRP2 translocator, and at least one 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.Described adult hepatic progenitor cell or stem cell also can have one, more than a kind of or all following feature: at least hematopoiesis mark CD45 and CD34 are negative, can be also that one or more for example CD105 of other hematopoiesis marks and HLA-DR are negative; Bile duct cell epithelium mark Ck19 (CK-19) feminine gender, more epithelium marks are negative possibly; 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 (AFP) of low expression level.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 there is one in the oval core of one or two kernel, more than a kind of or all.

Identify that by its CD (common determinant, " common determinant ") title specific cells surface molecular is as known in the art.Other names in this application or the use of title are as determined in this area.Also can find in an embodiment other explanations of concrete molecule.

Wherein in the time thinking that a cell is positive to special sign thing, this expression, when the suitable contrast of contrast is while suitably measuring, those skilled in the art will determine appearance or the sign of clear signal of this mark, as detectable in antibody or detect by reverse transcriptase polymerase chain reaction.Wherein said method allows this mark of quantitative evaluation, positive cell on average can produce the signal that is significantly different from contrast, as, but unrestricted, at least the more than 1.5 times of signal that compared with 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 are even higher.

Can use any suitable immunoassay cell-specific mark well known in the art, as flow cytometer, immunocytochemistry or affine absorption, Western engram analysis, ELISA etc., or by the technology of 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 can 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 alternatively further enrichment those show the cell (so this cell can be separated from described group) of specific characteristics (by means commonly known in the art).For example, rather than restriction, present the cell of one or more progenitor cells of the present invention or stem cell surface characterization of molecules, as, one or more marks listed above, can identify 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).Within any other method of enrichment of cell is included in invention scope.

Another aspect, the inventor thereby realized progenitor cell or the stem cell (vertebrates of a kind of novel separation from adult hepatic, preferably mammal, more preferably human cell) method, described progenitor cell or cells and characteristic of stem are at least one mesenchyme mark of coexpression (to being positive below), especially CD90, CD29, CD44, one in vimentin and α-smooth muscle actin (ASMA), more than one, as 2, 3, or 4 kinds or whole, and liver cell mark albumin (ALB) and one or more of other liver cell marks possibly.Described adult hepatic progenitor cell or stem cell also can have one, more than a kind of or all following feature: at least negative to hematopoiesis mark CD45, CD34 and CD117, and can also be negative to one or more other hematopoiesis marks; (CK-19) is negative for Ck19; Mesenchyme sample form, especially comprises with monolayer growth, flat pattern, obvious tenuigenin and/or have a kind of in the oval core of one or two kernel or all.

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 the liver source of separation.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), in above-mentioned embodiment (1) to (4), progenitor cell or the stem cell in the liver of any one source are also expressed CD29.In another embodiment (6), in above-mentioned embodiment (1) to (4), progenitor cell or the stem cell in the liver of any one source are also expressed alpha-fetoprotein.In another embodiment (7), in above-mentioned embodiment (1) to (4), progenitor cell or the stem cell in the liver of any one source are also expressed CD29 and alpha-fetoprotein.

In other embodiments (8), in above-mentioned embodiment (1) to (7), progenitor cell or the stem cell in the liver of any one source are CD45 and CD34 feminine gender.In other embodiments (9), in above-mentioned embodiment (1) to (7), progenitor cell or the stem cell in the liver of any one source are CD117 and Oct-4 feminine gender.In other embodiments (10), in above-mentioned embodiment (1) to (7), progenitor cell or the stem cell in the liver of any one source are CD45, CD34, CD117 and Oct-4 feminine gender.

In other embodiments (11), in above-mentioned embodiment (1) to (10), progenitor cell or the stem cell in the liver of any one source are CK19 feminine genders.In other embodiments (12), in above-mentioned embodiment (1) to (10), progenitor cell or the stem cell in the liver of any one source are CK7 feminine genders.In other embodiments (13), in above-mentioned embodiment (1) to (10), progenitor cell or the stem cell in the liver of any one source are CK19 and CK7 feminine gender.

In other embodiments (14), the progenitor cell in the liver of any one source or stem cell performance mesenchyme sample form in above-mentioned embodiment (1) to (13), especially comprise with monolayer growth, flat pattern, obvious tenuigenin and/or have the oval core of one or two kernel.

In another embodiment (15), in above-mentioned embodiment (1) to (14) progenitor cell in the liver of any one source or stem cell also express a kind of, more than molecules a kind of or all following character or the functions that are indicating liver cell sample: G6P, CYP1B1, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2.

In another embodiment (16), in above-mentioned embodiment (1) to (15) progenitor cell in the liver of any one source or stem cell also express one, more than a kind of or all following molecules: CD49e, CD13, CD54, I class major histocompatibility complex (MHC) are (HLA-ABC).

In another embodiment (17), in above-mentioned embodiment (1) to (16) progenitor cell in the liver of any one 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), progenitor cell or the stem cell low expression level alpha-fetoprotein (AFP) in the liver of any one source in above-mentioned embodiment (6) to (17).Preferably, the low-level expression level of measuring in normal liver cell that corresponds essentially to.The expression level of measuring in the human liver clone (as HepG2) of preferably, modifying lower than tumorigenesis.

Progenitor cell or the stem cell in the liver source that the present invention separates can preferably be shown cells and characteristic of stem, show especially in typical case at least limited (can be substantially unlimited) self, breed and undifferentiated ability.For example, rather than restriction, progenitor cell of the present invention or stem cell can breed goes down to posterity at least 4 times, as, go down to posterity at least 6 times, 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 or more.

Another aspect, the invention provides adult hepatic progenitor cell or the stem cell of the separation of or direct acquisition obtainable by method, clone and/or the cell mass that comprises 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, preferably vertebrates, Mammals and more preferably human subjects, to form primary cell group from described adult hepatic or its part; (b) primary cell group is inoculated in the matrix that is coated with type i collagen in WilliamsMedium E, wherein contain foetal calf serum, preferably 10% (V/V), EGF, preferably 25ng/ml, Regular Insulin, preferably 10 μ g/ml, dexamethasone, preferably 1 μ M; (c) allow cell to adhere to described matrix at least 24 hours from primary cell group, then matrix is replaced by the fresh matrix that contains composition in (b); (d) culturing cell two weeks (preferably 15 days) in matrix described in (c); (e) matrix is replaced by the DMEM containing high (concentration) glucose and FCS, preferably 10%, continue culturing cell, thereby progenitor cell of the present invention or stem cell occur and propagation; (f) optionally and preferably, make cell obtain approximately 70% converge and cell at least goes down to posterity once, preferably at least 2 times, wherein cell is seeded to matrix described in (b) and cultivates in matrix described in (e).

Step (a) can preferably include by allowing cell through at least one aperture sieve that is 0.25mm primary cell that dissociates, and is preferably decreased to gradually the sieve of 0.25mm by a series of meshes, as, in embodiment 1.

The matrix of step (e) can preferably not comprise dexamethasone, Regular Insulin and EGF in one embodiment.Do not comprise in another embodiment the somatomedin of any exogenous interpolation.

On the one hand, the invention still further relates to aforesaid method.

On the other hand, the invention provides adult hepatic progenitor cell or the stem cell of the separation of or direct acquisition obtainable according to the operating process of statement in embodiment 1, clone and/or the cell mass that comprises described adult hepatic progenitor cell or stem cell.

On the other hand, the inventor is by method of the present invention, especially as recorded in embodiment 1, adult people hepatic progenitor cell or stem cell cell mass (clone) are set up, and on February 20th, 2006, the clone of described separation is preserved in to 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 being provided by preservation people: ADHLSC).Therefore, an aspect of of the present present invention relates to and is preserved in cell, clone and the cell mass of the BCCM number of including for the separation of LMBP 6452CB (herein for " LMBP 6452CB " clone), subbreed comprises clone's subbreed, also relate to its offspring, break up offspring comprising it, especially liver cell or liver cell like cell prepared therefrom, also relates to the derivative of its genetic modification.

Those skilled in the art will know that progenitor cell or the stem cell that the method according to this invention obtains from adult human liver, the clone that comprises described progenitor cell or stem cell and cell mass can have biological property, especially consistent with the clone of above-mentioned preservation or similar propagation and differentiation capability, cellular form and/or marker expression, although they can have difference (due to the normal heritable variation between people) in heredity.Therefore, there is progenitor cell or stem cell or the cell mass of or similar biological property consistent with the cell mass of preservation, especially from liver, be also included within the scope of the invention.

Another aspect, the invention provides the progenitor cell of the adult hepatic that comprises separation or the cell mass of stem cell, and described cell or stem cell have above-mentioned feature, optionally through further modifying, as genetic modification.In one embodiment, described cell mass can comprise approximately 5% or more than, as, approximately 10% or above described progenitor cell or stem cell, approximately 20% or above, approximately 30% or above, approximately 40% or above, approximately 50% or above, approximately 60% or above, approximately 70% or above, approximately 80% or above or approximately 90% or above progenitor cell or stem cell, or and the group of described progenitor cell or the basic homogeneous of stem cell or homogeneous.

On the other hand, the invention provides by progenitor cell of the present invention or the stem cell in propagation liver source (optionally through further modifying, as genetic modification) and the clone set up.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 adult hepatic progenitor cell or the stem cell of the separation of the obtainable or direct acquisition of method (comprising its preferred embodiment) by the invention described above, clone and/or the cell mass that comprises described adult hepatic progenitor cell or stem cell.

In a specific embodiments, therefore the invention provides from the method (Fig. 1) of progenitor cell mesenchyme-sample stem cell of normal (people) liver isolating active of adult.Stem cell can breed in substratum, and the mark of expressing multiple liver cell type, for example, albumin (liver cell), vimentin (stellate cell), α-smooth muscle actin (ASMA) (Fig. 2 A).They do not have the phenotype of courage, as (Fig. 2 B) shown in negative Cyfra21-1 immunostaining and RT-PCR analysis.While utilizing flow cytometer to detect, ADHLSC is aobvious negative to CD45, CD34 and CD117, represents that it is not polluted by lymph hematopoietic lineage.By contrast, ADHLSC is to CD90, CD29 and CD44, and the mark of mesenchyme system is aobvious positive.

In one embodiment, described cell can keep its multiplication capacity after trypsinase/EDTA processes.In the matrix limiting, incubated cell allows these cell-specifics to be divided into liver cell (Fig. 3).According to other specific criteria, they cannot transdifferentiation scleroblast or adipocyte, as utilizes special energy (multipotent) people mesenchyme medullary cell viewed.The ability of its propagation, and their liver specificity increases the efficacy and saferry that liver cell is transplanted.In addition,, because it is derived from adult, these stem cells can avoid immunity, ethics and the carcinogenic problem that embryonic cell is relevant.

the differentiation of progenitor cell of the present invention or stem cell

Except detecting cell sign thing, the differentiation in vitro and/or in vivo of research cell can provide information.

The inventor also finds, the progenitor cell from liver or the stem cell that obtain by aforesaid method can have specificity differentiation capability.Particularly, stem cell can be divided into liver cell or liver cell like cell.In another embodiment, described cell is regardless of turning to mesoderm (mesenchyme) cell type, for example, as, osteocyte, chondrocyte, myocardial cell, phoirocyte, Tenocyte cell (tendonocyte), adipocyte or stroma cell (stromal cell).

The adult hepatic progenitor cell of separation 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 system) or its offspring, can advantageously be divided into cell, especially liver cell or the liver cell like cell of hepatic cell line through induction.This differentiation can occur in body external or in vitro.Therefore, the present invention also provides from the progenitor cell of the present invention's separation or stem cell and produces the method for liver cell or liver cell like cell, and liver cell or the liver cell like cell of acquisition are provided.

In a specific embodiments, therefore progenitor cell or the stem cell in the liver source that the present invention separates 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 be in vitro, observe in vitro or body towards the ability of certain specific cell type differentiation or lack this ability.By example, as known in the art, carry out evaluate differentiation by cell being exposed in the matrix containing specificity differentiation in vitro or in vitro.Otherwise, can be in vivo assess the differentiation (as, cell transplanting, injection or that use) of introduced (cell) by destiny subsequently.Those skilled in the art can identify the differentiation towards particular cell types by phenotype principle (including but not limited to expression and/or specific metabolic activity or other physiology paths of cellular form, protein marker).

Be divided under the condition that the cell of hepatocyte lineage can be advantageously exists in cytokine and somatomedin (can be liver-specific) and occur.For example, pHGF (HGF) or spreading factor (scatter factor) are the cytokine of known promotion to liver cell phenotypic differentiation.Similar, the non-limiting list 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.Inducing hepatocyte differentiation is being known in the art, and can further be optimized by technician.

Can identify by means commonly known in the art and separate from its undifferentiated counterpart (counterpart) subsequently the cell of differentiation.For example,, by identifying the cell being induced to differentiate making noble cells number exceed selectivity culturing cell under the condition of undifferentiated cell.Similarly, the morphological change not having by undifferentiated counterpart and feature are identified the cell of differentiation, as the complicacy of the size of cell, shape or the distribution of cell within a cell device.The method of the cell of the evaluation differentiation of also considering is by the expression of 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 the variation of gene expression response 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, genetic modification described above.The present invention also comprises the offspring of adult hepatic progenitor cell or stem cell, comprises and breaks up offspring.

In one embodiment, in order to improve the progenitor cell of the present invention of acquisition or the replication of stem cell, as known in the art, can be by cell telomere (telomerised).If the mode genetic modification that cell is recorded and translated at transit cell with TERT by the nucleic acid of the coding side human telomerase reverse transcriptase (TERT) of any species, cell is described to by " telomere ".This term is also for the offspring of the cell originally transformed, and it has inherited the ability with higher level expressing TERT coding region.TERT encoding sequence is taken from or is transformed the gene from Mammals TERT in typical case, in the following example as shown in the mankind and mouse TERT.Can be by the carrier genetically modified cell with suitable by its telomere, thus the composition (TERT) of telomerase catalytic expressed with higher level.Especially suitable is the human telomerase catalyst component (hTERT) providing as WO1998/14592.For some application, can use other TERT sequences.Also mention the additive method of cellular immortalization, as the DNA (US 5 with coding SV40 large T antigen, 869,243, WO 1997/32972), with Epstein Bar virus infection, introduce proto-oncogene if myc and/or ras, introducing virus replication gene are as adenovirus E 1 a and and have the immortalized cell line of expecting phenotype and merge cell is carried out to genetic modification.In the time that cell is used for the treatment of object, be conventionally not too applicable to proto-oncogene or tumour virus (oncovirus) product transfectional cell.

Conventionally; in the time considering described cell or its offspring (comprising the offspring of its differentiation) to be used for the treatment of; as this cell will be introduced into especially human body of human or animal, preferably no-go end granulation or other immortalization methods are transformed progenitor cell of the present invention or stem cell.

In the present invention, as substantially known in the field, before subsequent applications is as treatment or research, can or transform the progenitor cell from liver or stem cell or its offspring that obtain with object nucleic acid stability or transient transfection.Object nucleotide sequence can include but not limited to, as, those codings improve useful cell type in 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 carry out composing type or induced express polypeptide, described polypeptide is conventionally by liver cell especially liver cell expression, but in patient defect or disappearance.This indicia of defects patient's pathological state.Use after the cell of transformation like this can recoverin matter production, thereby contribute to treat patient.For example, progenitor cell or stem cell or its offspring can be contained coding metabolizable protein as ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, carbamyl phosphate synthetase, 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, P450 enzyme, and/or the allogeneic dna sequence DNA of ethanol dehydrogenase.As an alternative, the plasma proteins that cell can contain coding secretion is as the DNA of 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 in such a way on dissecting, and allows range protein efficiently to discharge into blood flow.Therefore, there is the particular cell types of the protein of systematicness effect with respect to common generation coding, if while being especially difficult to gene integration to these cells, described encoding gene is inserted in liver cell of the present invention.For example, various hormone genes or specific antibody gene can be inserted in liver cell of the present invention and secrete its gene product to the recycle system.

Use traditional gene transfer method that nucleic acid is introduced to 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 to cell comprises microinjection and electroporation.Chemical process is also the standard method of DNA being introduced to mammalian cell as calcium phosphate is total to-precipitates and DNA is mixed to liposome.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 (is shown in, as, Ausubel etc., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) and gene therapy has been developed with virus vector and successfully for clinical (see, as, Rosenberg, Deng, N.Engl.J.Med, 323:370 1990).

the purposes of progenitor cell of the present invention or stem cell

The progenitor cell of the present invention that is derived from liver separating or stem cell, the clone that comprises described progenitor cell or stem cell and/or cell mass can be used for different objects and (should know following purposes and apply and can extensively consider any adult hepatic progenitor cell defined above or stem cell, especially obtainable by method defined above or the cell of direct acquisition, the cell that shows above-mentioned restriction feature and LMBP 6452CB clone, preferably people's liver stem cells system (ADHLSC) in adult source; Its offspring; Or the derivative of its genetic modification), it includes but not limited to:

-use according to the progenitor cell of separation of the present invention or stem cell or its group, carry out liver cell transplanting in order to treat liver metabolism defect, liver degenerative disorders (liver degenerative disease) or fulminant hepatic failure (fulminant liver failure)

-use according to the progenitor cell of separation of the present invention or stem cell or its group and prepare biology-artificial liver device,

-prepare human liver disease's animal model by transplant progenitor cell or stem cell or its group of separating according to the present invention in animal,

-prepare in vitro toxicology, pharmacology animal model by the progenitor cell or stem cell or its group that separate according to the present invention,

-on the progenitor cell separating according to the present invention or stem cell or its group, test new drug, comprise the antiviral for human hepatitis virus.

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 mature hepatocytes (Figure 4 and 5).In addition, transplant in the transplanting mice serum after 10 weeks and human albumin detected at these, and Level of Alpha Fetoprotein do not detected.

Can be used for transplanting, the animal model of liver failure or the transplanting of human virus's hepatitis animal model of congenitalDy olism according to progenitor cell of the present invention or stem cell (will say especially, certainly include but not limited to LMBP 6452CB system).Therefore cell of the present invention can, for the treatment of liver related disease, include but not limited to the obstacle of liver failure, hepatitis, congenital metabolism.

In an exemplary, as shown in Example 1, the inventor has proved, when by intrasplenic injection as immunity-defect Mammals (more preferably rodent, especially mouse or rat) while 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 of progenitor cell of the present invention or stem cell will be contained, preferably from the mankind, (what will say especially is, yes but be not limited to LMBP6452CB system), introduce (as by injection) and allow to implant in the immune deficiency Mammals of heredity or chemical improvement, thereby obtaining animal model.Preferably, this population can comprise at least 2 × 10 ⁶individual cell of the present invention.Technician is by clear other modes of using cell mass of the present invention and inducing the described liver transplantation from adult hepatic progenitor cell or stem cell.

As described in detail in embodiment 1, the cell of transplanting does not have hyper-proliferative, thereby has underestimated its advantage in the mankind or other animals (preferred mammal) Transplanted cells.

The present invention also comprises and using according to the progenitor cell of separation of the present invention or stem cell or its group (will say especially, certainly include but not limited to LMBP 6452CB system) for following object:

-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 defects, familial hypercholesterolemia and other lipid metabolism disorders, urea cycle disorder, glycogenosis (glycogenosis), galactosemia, fructosemia (fructosemia), tyrosinemia (tyrosinemia), protein and carbohydrate metabolism defect, organic aciduria, mitochondrial disease, peroxysome and lysosome are not normal, protein synthesis is abnormal, liver cell translocator defect, glycosylation defect etc.,

-transplant and treat acquired liver degenerative disease according to hepatic progenitor cell of the present invention or stem cell,

-use according to hepatic progenitor cell of the present invention or stem cell and treat fulminant hepatic failure and acute or chronic liver failure,

-in bioartificial livers device and liver supplementary unit, use according to hepatic progenitor cell of the present invention or stem cell,

-by transplanting in small-sized and macrofauna according to human liver disease's animal model of hepatic progenitor cell of the present invention or stem cell acquisition,

-prepare the life history, propagation, resistance, result for the treatment of, purposes and any use that human hepatitis virus infection animal model (HBV, HAV, HCV, HEV, HDV...) carrys out Effect of Anti virus drugs according to the research of hepatic progenitor cell of the present invention or stem cell transplantation

-use and prepare in vitro 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,

-by insert the gene therapy of the virus sequence that can increase in vitro in progenitor cell according to the present invention or stem cell,

The animal model of-research people liver cell metabolism,

-by using according to progenitor cell of the present invention or stem cell, tolerance heterogenote, 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 offspring, is intended for treatment application, as, organizational project and cell therapy.

Those skilled in the art understand that purposes described in detail herein can comprise progenitor cell or stem cell, the clone that comprises 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 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 (optionally genetic modification), can be for cell replacement therapy.Can come supplementary functions sexual cell or replacement and lose to the destination organization dosed cells of object the cell of function.As an alternative, also can consider to provide the method for noble cells (especially liver cell or liver cell like cell), wherein under differentiation factor exists, make progenitor cell or differentiation of stem cells, separate and be applied to object.

The morbid state or the defect 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 induction 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, Caroli disease, crigler-najjar syndrome, fructosemia, galactosemia, glycosylated defect, other carbohydrate metabolism disturbances, carbohydrate deficiency (carbohydratedeficient), refsum disease and other peroxysome diseases, Niemann Pick disease, wolman disease and other lysosomal diseases, 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, thrombin defect, hemophilia B, pku, Wilson's disease (Wilson ' s Disease), fulminant hepatic failure, liver failure after hepatectomy, 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 system) or its offspring (comprise the offspring of differentiation, especially liver cell or liver cell like cell, optionally as detailed above through genetic modification) purposes that is used for the treatment of and/or for the preparation of the purposes of the medicine for the treatment of hepatic diseases.This disease can comprise the disease that affects hepatic tissue, and especially considering affects the disease of hepatocyte activity and/or function, and can represent as the impact of the impact of inborn error, morbid state, wound, toxic action, virus infection etc.Especially consider the hepatic diseases of listing in this explanation.Use according to cell of the present invention and can in object, cause reconstructed tissue or regeneration.To allow them to transplant or to move to the mode dosed cells of the tissue site of expectation, and the region of reconstruct or refresh function defect.

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 system) or its offspring, comprise the offspring of differentiation, especially liver cell or liver cell like cell, optionally it is by genetic modification.Normally use with treatment significant quantity, the part of expectation or the amount of systemic effect or performance are provided conventionally.

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, containing the clone of described 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 (comprise and break up offspring), especially liver cell or liver cell like cell, optionally 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 is (referring to Gupta etc., Seminars in LiverDisease 12:321, 1992), introportal infusion, liver pulp injection, under Glisson's capsule, abdominal injection or intrauterine injection enter embryo or fetus uses hepatic progenitor cell or the stem cell of separation of the present invention, containing the clone of described 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, can by hepatocyte transplantation (LCT) by the present invention be derived from the progenitor cell of liver or stem cell, containing the clone of described hepatic progenitor cell or stem cell or cell mass, (what will say especially is, include but not limited to LMBP 6452CB system) or its offspring (preferably by genetic modification) for organizational project and cell therapy.Liver cell is transplanted and liver stem cells transplanting (LSCT) refers to the mode that causes entering liver and cell implantation with any, preferably through portal vein, also can inject by direct liver, or by intrasplenic injection, inject the ripe liver cell of cell of the present invention or the technology of hepatic progenitor cell of comprising.

For example, after after separating or very low temperature are preserved and thawed, the form of the cell suspension that cell can be in any substratum provides, and preferably contains human albumin.

In one embodiment, the present invention considers to separate progenitor cell of the present invention or stem cell with patient's self hepatic tissue.This cell is the autologous property of patient, and easily uses to patient.In addition,, if patient has the hereditary defect that indicates particular pathologies condition, the cell obtaining by genetic manipulation can be avoided this defect.

In another embodiment, progenitor cell of the present invention or stem cell can be never to separate and obtain in patient's self tissue.In the time considering to use this cell to patient, 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 host is right).

Immunity system is distinguished the product that oneself and the ability of nonego depend on major histocompatibility complex (MHC) to a great extent, and its gene is on No. 6 karyomit(e)s 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 substantially 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 (but not tranquillization) T cell of B cell, scavenger cell, dendritic cell, Langerhans cell and activation.

Normally multiple alleles of HLA gene locus, 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 HLA gene locus is closely linked, HLA antigen can be also that conservative haplotype form exists.

Need cell therapy of the present invention object can by for whether exist anti-hla antibody and HLA genotype thereof and/or phenotype (as, on lymphocyte; As use serological method or hereditary DNA analysis) screen.The progenitor cell obtaining according to the present invention or stem cell or liver tissue source or its donor are tested its HLA phenotype and/or genotype in a typical case and are selected suitable tissue or the cell for using, it has the HLA haplotype consistent with patient, or there is most HLA antigen allelotrope common in patient, and do not have or the HLA antigen of the minimum anti-hla antibody for being pre-existing in patient.The probability that transplanted cells is successfully accepted improves the increase of the HLA antigen number along with identical.Those skilled in the art will know that other variations of these considerations.

It is also conceivable that and obtain other approach that represented 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-autologous), conventionally use the immunosuppressant therapy of following, as used immunosuppressor, as ciclosporin or tacrolimus (tacrolimus) (FK506).As an alternative, described cell can be wrapped in and allows 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 autologous, or as described in performance HLA coupling approach.

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, the xenotransplant, the bioartificial livers device that contain non-human animal or non-human mammal cell).For example, rather than restriction, can be from pig for the particularly suitable non-human mammal cell of human treatment.

About a problem of the therepic use of 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 and be followed by using first of subsequent applications; And can be determined by disclosure one of ordinary skill in the art.In typical case, application dosage or dosage will provide the cell for the treatment of significant quantity, reach local and systemic effect and the performance of expection.

The human research of current autologous property single core medullary cell, experience dosage from 1 to 4 × 10 ⁷individual cell not etc., has not obtained challenging achievement.But different situations may need to optimize the amount of dosed cells.Therefore the cell of, using is different and different with the object of processing.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, approximately 5 × 10 ⁵, approximately 1 × 10 ⁶, approximately 5 × 10 ⁶, approximately 1 × 10 ⁷, or approximately 2 × 10 ⁷, approximately 3 × 10 ⁷, approximately 4 × 10 ⁷, approximately 5 × 10 ⁷, approximately 6 × 10 ⁷, approximately 7 × 10 ⁷, approximately 8 × 10 ⁷, approximately 9 × 10 ⁷, or approximately 1 × 10 ⁸individual cell can be applied to human subjects.But, accurately determine the individual factors that treatment effective dose can be based on each patient, comprise the time length after its stature, age, tissue injury size and damage occur, can be determined easily according to the disclosure and this area general knowledge by those skilled in the art.

Preferably, for the cell mass purity that comprises progenitor cell of the present invention or stem cell used can approximately 50 to approximately between 55%, approximately 55 to approximately between 60%, approximately 65 to approximately between 70%.More preferably purity can approximately 70 to approximately between 75%, approximately 75 to approximately between 80%, approximately 80 to approximately between 85%; Most preferably purity can approximately 85 to approximately between 90%, approximately 90 to approximately between 95%, approximately 95 to approximately between 100%; Can according to compose as cell surface marker in cell mass determine as described in stem cell purity.Dosage can easily be adjusted (as needed to increase dosage compared with low-purity) by those skilled in the art.

Those skilled in the art can easily determine the cell concentration in composition to be administered and optional additive, vehicle and/or carrier in the inventive method.In typical case, any additive (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 milligram rank, 0.0001 to approximately 5% (w/w or w/v) according to appointment, preferred approximately 0.0001 to approximately 1%, most preferably from about 0.0001 to approximately 0.05% or approximately 0.001 to approximately 20%, preferably approximately 0.01 to approximately 10%, and most preferably from about 0.05 to approximately 5%.

In the time using therapeutic composition of the present invention, be conventionally mixed with unitary dose injection form (as, solution, suspension, dispersion agent, emulsion).The pharmaceutical dosage form that is suitable for injection comprises aseptic aqueous solution and dispersion agent.As used herein, solution or dispersion agent comprise cell of the present invention and still keep therein active pharmaceutically acceptable carrier or thinner.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 antibacterial and anti-mycotic agents, for example, parabens, butylene-chlorohydrin, phenol, Sorbic Acid etc. guarantee to prevent microbial activities.

Under many circumstances, wish to contain isotonic agent and guarantee the activity of cell, for example, sugar, sodium-chlor etc.The desirable isotonicity of the present composition can be by realizing with 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 containing the damping fluid of sodium ion.

In the time introducing the offspring of target progenitor cell or stem cell or differentiation to the object that has demand, in order effectively to increase the survival of cell, possibly described cell is mixed in biopolymer or synthetic polymer.The example that is applicable 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 extend the absorption of injectable medicine type with slowly-releasing absorption agent, for example, aluminum monostearate and gelatin.According to the present invention, but carrier, thinner or the additive of any use be necessary and progenitor cell or stem cell compatibility all.

Can be by preparing aseptic injectable solution by implementing cell used time of the present invention and mixing with other compositions (if required) of various amounts in the suitable solvent of aequum.

This composition also can be mixed with suitable carrier, 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 reference standard text, as " Remington ' s pharmaceutical science " the 17th edition, 1985 by reference to being incorporated to herein, does suitable preparation, and avoid unnecessary experiment.

If necessary, can use pharmaceutically acceptable thickening material that the viscosity of composition is maintained to selected level.Preferable methyl Mierocrystalline cellulose, because it is easy to commercial acquisition and convenient operation.Other suitable thickening materials comprise, for example, and xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, carbomer etc.The concentration of preferred thickening material depends on selected reagent.In the time using certain amount, under this amount, selected viscosity will be obtained.Conventionally prepare viscous composition by add this thickening material in solution.

other purposes of progenitor cell of the present invention or stem cell

(what will say especially is for the clone of having considered progenitor cell of the present invention or stem cell below, contain described dirty progenitor cell or stem cell or cell mass, include but not limited to, LMBP 6452CB system) or its offspring (comprise the offspring of differentiation, especially liver cell or liver cell like cell, the optionally 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 the cell response (as toxicity) to bioactive agents (biological agent or pharmacologic agent), comprise and make the derivative of cell culture or its differentiation contact one or more of biologies or pharmacologic agent, identify the one or more of cell responses to one or more of biologies or pharmacologic agent, relatively the cell response of cell culture and the cell response of control cultures.This reaction can determine by detection molecules is active, such as, but not limited to, the enzyme of alkaline phosphatase, Cytochrome P450, urea approach etc.

In addition, for example, can be by offspring, the especially liver cell of progenitor cell of the present invention or stem cell or its differentiation or liver cell like cell, screen cytokine, chemokine, pharmaceutical composition and somatomedin, more clearly to illustrate its differentiation to 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 destination organization and the tissue engineering devices of cytokine, somatomedin or differentiation factor optionally, wherein cell of the present invention is used for producing tissue, especially liver organization, especially comprises hepatocellular tissue.Engineered organ can coordinate biocompatible scaffold to use, and described support sustenticular cell is with three-dimensional conformation (structure) growth, and support is biodegradable.The implantable object that need to change organ, its part or specific part of engineered organ producing from stem cell of the present invention.The present invention has also designed the cell of stem cell or its differentiation as the purposes of bio-reactor (as liver supplementary unit).

Host can be implanted in organ from stem cell of the present invention, its part or region.Transplanting can be autologous, is the acceptor of engineering tissue from the donor of the stem cell of organ or organ unit.Transplanting can be allos, is not the acceptor of engineering tissue from the donor of the stem cell of organ or organ unit.Once be transferred to host, engineered organ can reappear the function and structure of natural host tissue.Engineered organ will be conducive to the multiple application of theme of the present invention, the damage that comprises treatment cancer and other diseases disclosed herein, birth defect or cause due to surgical excision.

As the instrument of drug abuse test and performance history, liver cell and offspring thereof can be used for assessing drug-induced gene expression pattern to be developed and change.The gene expression pattern that potential drug causes changes and can compare with the drug-induced variation of those known effect livers.This will can screen on compound drugmaker in early days on the impact of liver in exploitation, thereby saved time and money.The whole pedigree of liver cell, from progenitor cell to mature cell, also can be used for the toxicity of testing drug to liver, how metabolism of drugs.At present, drugmaker has any problem in the conforming liver cell supply side for toxotest of acquisition.The inventive method and cell have met this demand.

In addition, adult hepatic progenitor cell of the present invention or stem cell or its offspring (comprise and break up 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 be used collagen, lectin, ln or fibronectin processing, for adherent cell or stay undressed.Pour into body fluid according to known program by device and carry out detoxification, and then defeated time patient.The example of the LAD of an applicable cell of the present invention has been described in International Patent Publication PCT US00/15524.

Adult hepatic progenitor cell of the present invention or stem cell or its offspring can break up in vitro and substitute mature hepatocytes in " ADMET " (use, distribution, metabolism, elimination and toxicology) or cytotoxicity test.

Break up by adult hepatic progenitor cell of the present invention or stem cell or its offspring the liver cell or the liver cell like cell that obtain and can provide a kind of for studying liver development, liver cell metabolism or the biological external model of liver cell; Be used for screening differentiation, propagation or toxicity molecule.Also consider the genetic manipulation of this cell, they can be for research liver development, 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

Liver cell separation method

Obtain human liver cell from the whole liver from healthy corpse or Lungs from Non-Heart-Beating donor or liver part.After total Xining (as, after total Xining 6 to 12 hours) isolated cell, until before perfusion liver all in University of Wisconsin's medium (the University of Wisconsinmedium) be stored on ice.Use classical two step perfusion technique isolating hepatocytes { Seglen, 1976}{Stephenne, 2005}.Via EGTA solution for obvious blood vessel, (Earl balanced salt solution, containing Ca subsequently for liver organization ⁺⁺and Mg ⁺⁺, 0.5mM EGTA, 5mM Hepes, 2mg/l gentamicin and 100,000IU/I penicillin G) and digestive ferment solution at 37 ℃, pour into separately 9 to 12 minutes.(EBSS is containing Ca for digestion solution ⁺⁺and Mg ⁺⁺, 5mM Hepes, 2mg/l gentamicin and 100,000IU/I penicillin G) Trypsin inhibitor SBTI that contains 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 clean 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

Single cell suspension is resuspended in the Williams ' E matrix that is supplemented with 10% foetal calf serum (FCS) (Perbio, Hyclone), 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 is inoculated in I type mouse tail collagen (BD Biosciences) coated shaking flask or plate (as, 6 orifice plates) (Greiner Bio-one), and containing 5%CO ₂in water saturated environment, at 37 ℃, cultivate.After 24 hours, change substratum and remove the cell not adhering to, within after this every 3 days, upgrade.Within fortnight, microscopic examination every day culture, analyzes substratum for every three days.Then substratum is changed into and there is the DMEM (Invitrogen) of (Perbio, Hyclone) and 1%P/S (Invitrogen) that high glucose concentration is supplemented with 10%FCS to accelerate adult hepatocellular removal.Confirm to there is cell type spontaneous appearance the subsequently of mesenchyme sample form through phase microscope, breed and fill up the blank space of orifice plate.Cultivate between 15 to 20 days and occur these cells, and present flat pattern, obvious tenuigenin and/or have the former forming core of ovum (Fig. 1) of one or two kernel.When reaching 70% while converging, peel off cell with 0.25% trypsinase and 1mM EDTA, and to expect concentration renewed vaccination.The cell suspension analysis of carrying out with the flow cytometer cell mass after 2 times that shows to go down to posterity becomes homogenization.For go down to posterity at every turn, also can carry out analysis of cells suspension by RT-PCR and immunofluorescence.

Cell characteristics

For fear of may be by other types cell contamination, carry out immunocytochemistry and study the phenotype of these cells.Because they derive from liver, in the fixing cell of paraformaldehyde, analyze Specific marker as albuminous expression.As shown in Figure 2, the albumin that uses mono-clonal (Sigma clones HAS-111) or polyclone (Chemicon) antibody test only to express in liver cell.Also evaluate abreast the expression of mesenchymal cell mark, shown that these cells are vimentin and α smooth muscle actin immunity positive (Fig. 2).In the research of 7 times of going down to posterity, its phenotypic characteristic has very large stability up to now.

Cytodifferentiation:

With 0.5-1 × 10 ⁴/ cm ²density cell be seeded in the DMEM that is supplemented with FCS and P/S, be coated with on 6 coated orifice plates of I type mouse tail collagen.After 24 hours, substratum is changed into Iscove ' smodified Dulbecco ' s substratum (IMDM) (Invitrogen).For induction, cell is hatched 2 weeks in the inducing culture that contains IMDM supplementary 20ng/ml HGF (Biosource), 10ng/ml bFGF (Peprotech) and 0.61g/l niacinamide (Sigma).After this, cell is hatched in supplementing 20ng/ml oncostatin M (Sigma), 1 μ M dexamethasone (Sigma), 50mg/ml ITS (Regular Insulin, Transferrins,iron complexes, selenium) maturation medium (Invitrogen) containing IMDM.For induction and maturing step, within every 3 days, change and analyze substratum.After contacting with above mixture (cocktail), cell starts to lose its sharpened edge, shrinks and lose its initial form and present polygonal shape (Fig. 3) gradually.

Flow cytometry

With 1200rpm collecting cell after centrifugal 5 minutes, and with the concentration of 500 to 1000 cell/μ l, cell is resuspended in PBS.Then at 4 ℃, hatch 30 minutes with antibody.Corresponding control antibodies hypotype is for evaluating the non-specific binding of monoclonal antibody.Then rinsing cell, is resuspended in (Beckham Coulter) reads with Beckham Coulter flow cytometer.

RT-PCR

According to the specification sheets of manufacturers, use TriPure separation agent (Poche) from grow in the cell 6 orifice plates, to extract total RNA, with reverse transcription test kit generation cDNA.In 25 μ l final volume, carry out pcr amplification with polysaccharase extending enzyme and suitable primer.After this, sample carries out electrophoresis and observes nucleic acid by ethidium bromide staining on 1% sepharose.

Immunofluorescence

Immunostaining, at room temperature, cell by 4% paraformaldehyde (V/V) fixed growth on the 12mm circular lid glass sheet of I type mouse tail collagen-coated 15 minutes, after this uses the 1%TBS solution (V/V) of Triton X 100 by cell permeabilization (Permeabilized) 15 minutes (Tris-HCl 50mM, NaCl 150mM, pH 7.4).Within 1 hour, prevent non-specific immunity dyeing by hatching at 37 ℃ in the TBS solution that contains 3% skim-milk.Cell is at room temperature hatched 1 hour with primary antibodie subsequently in same solution, with TBS rinsing 5 times, resists and hatches together 1 hour (1/500) with two.With core dyestuff DAPI (1/5000) to core carry out 30 minutes dyeing.After rinsing 3 times, prepared product is placed in to Fluoprep medium (BioMerieux, Brussels, Belgium) also with being furnished with CCD camera (T.I.L.L.photonics, Martin's Si Reed, Germany) Olympus 1 × 70 inverted microscope inspection.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 in conjunction with TILLvision software and obtain digital image.

The Molecular Detection of progenitor cell of the present invention or stem cell

Use aforesaid method, this embodiment for setting up the experiment of cell, set up the express spectra (ADHLSC) of following various kinds of cell mark:

The CD90 positive.CD90 or Thy-1, a kind of cell surface protein, is regarded as the mark that mesenchyme is.

The CD44 positive.CD44 is a kind of cell adhesion molecule and is used for identifying at least interstital stem cell of some type (MSC).

The vimentin positive.Vimentin is the III type median fiber that often can detect in a kind of mesenchymal cell and inoblast.

The albumin positive.Albumin is a kind of plasma proteins that is produced and secreted by liver.In liver cell, conventionally find that albumin is a kind of cytoplasm protein.

The CD29 positive.CD29, also referred to as integrin beta-1, is a kind of transmembrane glycoprotein, is also present in hepatic tissue, is considered to form together with integrin alpha participate in and the interactional functional receptor mixture of extracellular matrix.

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-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 film heterodimer.

The alpha-fetoprotein of low expression level.Alpha-fetoprotein is in a kind of primitive endoderm (primitiveendoderm) growth course and whole maturation, to express, reflect the protein of entoderm pedigree.High level expression alpha-fetoprotein conventionally discloses tumorigenicity and separates (tumorigenic shunt).

α-1 antitrypsin the positive.α-1 antitrypsin is the synthetic plasma proteins of a kind of liver.

Glucose 6-Phosphoric acid esterase (G6P) positive.G6P is a kind of liver enzyme that glucose 6-phosphoric acid hydrolysis is become to glucose and inorganic phosphorus, 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 in far-ranging structure.

Cytochrome P450 3A4 (CYP3A4) positive.CYP3A4 is a kind of key enzyme that participates in heterobiotin metabolism.

Hepatocyte nuclear factor 4 (HNF-4) positive.HNF4, a kind of nuclear receptor, is a kind of transcription factor that participates in energy metabolism regulation.

Tryptophane 2,3-dioxygenase (TDO) positive.TDO participates in first enzyme that tryptophane is oxidized in liver.

Tyrosine aminotransferase (TAT) positive.TAT is a kind of amino acid metabolism and gluconeogenetic plastosome liver specificity enzyme of participating in.

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.

CK8 (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 cell to export to from liver cell biliary tract.

Glutamate transporter 2 (EAAT2) positive.

Occur that above-mentioned point subrepresentation ADHLSC clone and the liver phenotype that much may participate in liver function and metabolism has close ties.

CD117 feminine gender, CD117, also referred to as c-kit, is the cell surface receptor of identifying HSC and MSC in a kind of medullary cell type, thereby characterizes undifferentiated stem cell to a great extent.

CD34 feminine gender.CD34 is the cell surface protein on a kind of medullary cell, indicates HSC and endothelial progenitor cells.

CD45 feminine gender.CD45, also referred to as human leucocyte antigen, is the tyrosine phosphatase that a kind of hematopoietic lineage cell (comprising hemopoietic stem cell) is expressed.

CD105 feminine gender.CD105, also referred to as SH2 or endoglin, is a kind of adhesion molecule.It is also considered to a kind of mescenchymal stem cell mark.

CD133 feminine gender.CD133 is a kind of hemopoietic stem cell mark.

HLA-DR feminine gender.These II class major histocompatibility complex antigens are film heterodimers of the restricted expression of antigen presenting cell.

Oct-4 feminine gender.Oct-4 is a kind of transcription factor of only being expressed by multipotential stem cell, and is that to maintain undifferentiated state necessary.

Cyfra21-1 (CK19) feminine gender.CK19 is widely used as the mark that biliary tract cell is bile duct cell.

Cytochrome P 2B6 (CYP2B6) feminine gender.In CYP2B6 participates in-and heterobiotin metabolism.

CD54: intercellular adhesion molecule-1 (ICAM-1), a kind of membrane glycoprotein.

Be not intended to limit by any way, according to it, the knowledge to cell sign thing has proposed the following explanation possible to above-mentioned data to the inventor: 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).Detect and exist albumin to come down hard upon the possibility of being polluted by stellate cell by immunofluorescence and RT-PCR.ADHLSC seems neither multipotency does not break up mescenchymal stem cell (CD45 feminine gender, CD34 feminine gender, CD117 feminine gender, Oct-4 feminine gender), neither liver stellate cell (the albumin positive).ADHLSC pedigree consistent with 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, with a kind of but be not the mode of restriction, be characterized as being the mescenchymal stem cell system with liver cell progenitor cell feature.

The transplanting of uPA-SCID mouse, histology and immunohistochemistry

1000000 ADHLSC (>=90% activity) are expelled to 6-to 14 age in days uPA ^{+ /+}the spleen of-SCID mouse.Before transplanting, mouse does not show detectable serum albumin.The immunohistochemical analysis of carrying out mouse liver sample on the thick liver section of 4 μ m-of phenodin and eosin (HE) dyeing is for the histopathologic overall evaluation.For immunostaining, under room temperature with first antibody by liver section overnight incubation.After hatching section with the polymkeric substance of peroxidase labelling and substrate chromogenic reagent, detect (Envision-DAB system, Dako, Carpinteria, CA).Use haematoxylin redyeing.

Transgene mouse model for this object combines with the hepatic pathology (uPA) with immunodeficient disease (SCID).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 mature hepatocytes (Fig. 5).In addition, transplant in the transplanting mice serum after 10 weeks and human serum albumin detected at these, the Level of Alpha Fetoprotein (mark that tumour is educated) of expression still can't detect.

The cell of transplanting as shown in microscopic examination does not have hyper-proliferative, and representing does not have the expression level of tumorigenesis colony and tumorigenesis mark alpha-fetoprotein and Ki67 normal.Normal expression level is equivalent to the expression level of measuring in normal liver cell substantially, and is as the expression level of measuring in HepG2 lower than people's liver cell of tumorigenicity transformation.

Embodiment 2

One is derived from 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 (optionally by genetic modification) exemplary process can be as follows.

Inject cell by continuous injection, be preferably no more than 25 to 50 × 10 ⁶individual cell/kg, preferred interval 4 hours, or interval 8 hours, or exceed 8 hours until 1 week, or exceed 1 week.Continue a few days injection 250 × 10 ⁶individual cell/kg, or 500 × 10 ⁶total cell concentration of individual cell/kg, preferably one week or preferably two weeks.Repeat if required 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 or through Port-a-cath R device or be inserted into any pylic blood vessel (preferably inferior mesenteric vein or colic vein) that is drained to through Broviac R device via surgery and enter portal vein by direct puncture.Conduit can be retained a few hours in position, preferably a couple of days, preferably several weeks, or preferably the several months until 2 years, or preferably the longer time in case the time of needs repeat infusion.

Immunosuppression starts from infusion same day, preferably the day before yesterday, preferably uses tacrolimus (FK506) and steroid.The preferred initial period 8ng/ml of blood level of tacrolimus, 6ng/ml after 3 months, 4ng/ml after 6 months, then remains on 4ng/ml left and right.Steroid preferably gives prednisone or Ultracortene-H, and preferably the 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, then in the time of 3 months, gradually reduce 0.25mg/kg, and stopped in the time of 6 months.Alternative immunosuppression can comprise, (use) alone or in combination ciclosporin A, anti IL2 receptor antibody, antithymocyte globulin or any anti-human lymphocytic polyclone or monoclonal antibody, mycophenlate mofetil mofetyl or azathioprine or any antimetabolite, ciclosporin, rapamycin (rapamune) or any other neurocalcin (calcineurin) inhibitor.

Claims (31)

1. the people's progenitor cell or the stem cell that derive from the separation of the adult liver of people, is characterized in that

(a) it expresses mark CD90, CD73, CD44, vimentin and α-smooth muscle actin;

(b) it expresses ALB; With

(c) it is negative to CK-19.

2. people's progenitor cell or the stem cell of the separation of claim 1, it also expresses one or more other liver or liver cell mark, and it is selected from CD29, alpha-fetoprotein (AFP), α-1 antitrypsin, HNF-4 and MRP2 translocator.

3. people's progenitor cell or the stem cell of the separation of claim 2, it expresses CD29, AFP, α-1 antitrypsin and MRP2 translocator.

4. derive from people's progenitor cell or the stem cell of the separation of people's adult hepatic, it is positive to CD90, CD29 and CD44, and be the albumin positive, the vimentin positive and α-smooth muscle actin positive, and be CD45, CD34, CD117 and CK19 feminine gender.

5. people's progenitor cell or the stem cell of the separation of any one in claim 1 to 4, wherein said cell has mesenchyme sample form, comprises following any one or all: monolayer growth, flat pattern, wide tenuigenin and have the avette nucleus of one or two kernels.

6. people's progenitor cell or the stem cell of the separation of any one in claim 1 to 4, wherein said cell can be divided into liver cell or liver cell like cell, and regardless of changing into mesoblastema type.

7. clone or cell mass, the hepatic progenitor cell that it comprises the separation described in any one in claim 1 to 6 or stem cell.

8. the people's adult hepatic progenitor cell or stem cell and the clone that separate, be deposited in Belgian microorganism cooperation preservation center (BCCM) on February 20th, 2006 according to budapest treaty, and its preserving number is LMBP6452CB, and subbreed.

9. people's adult hepatic progenitor cell of the separation of claim 8 or stem cell and clone, wherein said subbreed is clone's subbreed.

10. the method for the cell mass that obtains people's progenitor cell of separating described in any one in claim 1-6 or stem cell or comprise described progenitor cell or stem cell, the method comprises: (a) by the substrate that can adhere to cell from the primary cell group bed board of the fast adult hepatic of institute or its part formation by dissociating people's adult hepatic or its part, (b) will continue to cultivate at least 7 days from the described suprabasil cell of adhering to of this primary cell group, and (c) by the passage of step (b) at least one times.

The method of 11. claims 10, wherein will continue to cultivate at least 10 days, at least 13 days or at least 15 days from the described suprabasil cell of adhering to of this primary cell group.

The method of 12. claims 10, wherein by the passage at least twice of step (c).

The 13. people's adult hepatic progenitor cells that separate or stem cell, its clone and/or the cell mass that comprises described cell, it can obtain or directly be obtained by described method by the method for claim 10.

People's adult hepatic progenitor cell of the separation of 14. claims 13 or stem cell, its clone and/or the cell mass that comprises described cell, wherein said method comprises: (a) from people's object dissociate adult hepatic or its part, to form primary cell group from the fast adult hepatic of institute or its part; (b) by described primary cell group bed board to the substrate of the coated type i collagen in Williams Medium E substratum, described substratum comprises foetal calf serum, EGF, Regular Insulin and dexamethasone; (c) continue to make for 24 hours cell adhesion from described primary cell group to described substrate, subsequently substratum is replaced by the fresh culture with composition in (b); (d) in two time-of-weeks in substratum described in (c) culturing cell; (e) substratum is replaced by the DMEM that comprises high glucose and FCS, continues culturing cell, progenitor cell of the present invention or stem cell occur and breed thus; (f) make cell reach approximately 70% and converge, by passage at least one times, in the substratum of wherein said cell bed board to the substrate in (b) and in (e), cultivate.

People's adult hepatic progenitor cell of the separation of 15. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, the foetal calf serum that wherein (b) described substratum comprises 10% (v/v).

People's adult hepatic progenitor cell of the separation of 16. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, the EGF that wherein (b) described substratum comprises 25ng/ml.

People's adult hepatic progenitor cell of the separation of 17. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, the Regular Insulin that wherein (b) described substratum comprises 10 μ g/ml.

People's adult hepatic progenitor cell of the separation of 18. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, the dexamethasone that wherein (b) described substratum comprises 1 μ M.

People's adult hepatic progenitor cell of the separation of 19. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, wherein (e) described substratum comprises 10% FCS.

People's adult hepatic progenitor cell of the separation of 20. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, wherein (d) in 15 days in substratum described in (c) culturing cell.

People's adult hepatic progenitor cell of the separation of 21. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, wherein in (f) by passage at least twice.

People's adult hepatic progenitor cell of the separation of 22. claims 14 or stem cell, its clone and/or the cell mass that comprises described cell, wherein (a) dissociates by two step collagenase method.

23. 1 kinds of compositions, it comprises clone or the cell mass described in any one in the people's hepatic progenitor cell described in any one in claim 1 to 6,8-9,13,14-22 or stem cell or claim 7,8-9,13,14-22.

Clone or cell mass in people's hepatic progenitor cell in 24. claims 1 to 6,8-9,13,14-22 described in any one or stem cell or claim 7,8-9,13,14-22 described in any one, it is used for the treatment of.

25. 1 kinds of pharmaceutical compositions, it comprises clone or cell mass and pharmaceutically acceptable carrier described in any one in the people's hepatic progenitor cell described in any one in claim 1 to 6,8-9,13,14-22 or stem cell or claim 7,8-9,13,14-22.

26. implement the method for in vitro toxicity test, and it comprises:

Make to test clone or cell mass described in any one in the people's hepatic progenitor cell described in any one in medicament contact claim 1 to 6,8-9,13,14-22 or stem cell or claim 7,8-9,13,14-22, and if some words are observed at least one impact on described liver cell group of this test medicament.

The method of 27. claims 26, wherein said at least one impact comprises cell survival, cell function or the impact on the two.

28. implement the method for external drug metabolism study, it comprises: (i) make to test clone or cell mass described in any one in the people's hepatic progenitor cell described in any one in medicament contact claim 1 to 6,8-9,13,14-22 or stem cell or claim 7,8-9,13,14-22, (ii) if any, after the presumptive test time, observe at least one variation relevant with this test medicament.

The method of 29. claims 28, wherein said at least one variation comprises that described test medicament is in structure, concentration or the variation of the two.

30. liver supplementary units, it comprises clone or the cell mass described in any one in the people's hepatic progenitor cell described in any one in shell and claim 1 to 6,8-9,13,14-22 or stem cell or claim 7,8-9,13,14-22, and wherein said cell or cell mass are contained in described shell.

Clone in people's hepatic progenitor cell in 31. claims 1 to 6,8-9,13,14-22 described in any one or stem cell or claim 7,8-9,13,14-22 described in any one or the cell mass purposes in the medicine for the preparation of enhancing damage or ill liver regeneration.

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