CN1744823A - Engineering three-dimensional tissue structures using differentiating embryonic stem cells - Google Patents

Abstract

A method of producing a tissue engineering construct. The method includes providing a population of embryonic stem cells, seeding the embryonic stem cells on a cell support matrix, and exposing the embryonic stem cells to at least one agent selected to promote differentiation of the stem cells along a predetermined cell lineage or into a specific cell type. The step of exposing may be performed before or after the step of seeding.

Description

Use the differentiating embryonic stem cells engineering three-dimensional tissue structures

The application's case advocates to have precedence over the temporary patent application case the 60/432nd of application on December 10th, 2002, the file number of No. the 60/443rd, 926, the temporary patent application case of application on January 31st, No. 228 1 and application on December 9th, 2003 is the right of the patent application case of 0492611-0530.

Technical field

The present invention relates to use differentiating embryonic stem cells to produce engineering three-dimensional tissue structures.

Background technology

The embryonic stem cell (ES) that comprises hESC (hES) is owing to it becomes the resource 1-3 that is hopeful to be used for cell transplantation, 4 in the unique ability that the experience differentiation phase can produce various somatic cell pedigrees.Can induce the ES differentiation the suspension by cell is taken out and it is grown in from its feeder layer, this can cause cell aggregation and embryoid (EB) to form, and wherein continuous differentiation step 5 can take place.Several studies shows, directly provide by growth factor or can induce the ES cell differentiation to become particular lineage 6-9 by the chemical inducement that feeder cells provide indirectly.Yet these researchs all fail control ES cell differentiation to form complex organization.In some cell types, comprise that the physics inducement of surface interaction, shear stress and mechanical strain can have been induced differentiation 10-13.

Therefore, the expectation exploitation can promote the ES cell differentiation to become the method for engineering three-dimensional tissue structures.

Summary of the invention

On the one hand, the invention provides organizational project structure, it comprises that embryonic stem cell, can resist the three-dimensional cell carrier matrix of the convergent force that is applied by stem cell and at least a selected growth factor to promote stem cell along a predetermined cell lineage differentiation or promote that stem cell is divided into a particular cell types.Stem cell can be mammalian embryonic stem cell, for example hESC.Cell carrier matrix can comprise a kind of (lactic acid)-poly-(lactic acid-be total to-glycolic) mixture, for example 50/50 mixture of one poly-(L-lactic acid) and poly-(lactic acid-be total to-glycolic) of gathering.

Under a convergent force that is applied by embryonic stem cell, the matrix cross-sectional area can reduce 50% at the most, for example, reduces 40%, 30%, 20%, 10% or 1% at the most.Described cell carrier matrix can further comprise a coating, and this coating comprises that one can promote the reagent of cell adhesion, for example, can promote fibronectin, integration element or the oligonucleotides of cell adhesion.Described cell carrier matrix can be biodegradable or not biodegradable matrix.

This organizational project structure can further comprise one or more biomolecule that place cell carrier matrix, little molecule or bioactivator.This organizational project structure can further comprise the gel of coating cell carrier matrix surfaces externally and internally.The exemplary gel comprises the matrigel (Growth Factor Reduced Matrigel) of collagen gel, alginates, agar and minimizing growth factor.Gel can further comprise one or more in laminin, fibrin, fibronectin, proteoglycan, glycoprotein, glucosaminoglycan, chemotactic factor (CF) or the growth factor (for example, cell factor, eicosanoid or differentiation factor).

On the other hand, the invention provides a kind of method of generation one organizational project structure.Described method comprises provides an embryonic stem cell colony, described embryonic stem cell is seeded on the cell carrier matrix and described embryonic stem cell is exposed at least a selected agent to promote stem cell along a predetermined cell lineage differentiation or promote that stem cell is divided into a particular cell types.Exposing step can carried out before or after the inoculation step and can carry out in a serum free medium.This cell carrier matrix can be three-dimensional and the available one reagent coating that can promote cell adhesion.Embryonic stem cell can be placed a gel, and embryonic stem cell is inoculated in comprises on the cell carrier matrix described gel is placed on the surfaces externally and internally of cell carrier matrix.

Described agent can be a growth factor, a mechanical force, a voltage, a biological activating agent, a biological molecule, a little molecule or its a certain combination.Described mechanical force can comprise a circumference stress, a shear stress, a liquid static stress, a compression, a tensile stress or its any combination.As the part that step is provided, can in the presence of a growth factor, cultivate embryonic stem cell.

Definition

" biomolecule ": term used herein " biomolecule " refer to generally to be found in cell and the tissue, no matter be natural existence or artificial (for example the manufacturing, by synthetic or recombination method) molecule (for example, protein, amino acid, peptide, polynucleotides, nucleotide, carbohydrate, sugar, fat, nucleoprotein, glycoprotein, lipoprotein, steroids, or the like).The concrete kind of biomolecule includes but not limited to enzyme, acceptor, neurotransmitter, hormone, cell factor, cell response conditioning agent (such as growth factor and chemotactic factor (CF)), antibody, vaccine, hapten, toxin, interferon, ribozyme, antisense agent, plasmid, DNA and RNA.

" biocompatible ": term used herein " biocompatible " is intended to be described in and can cause that in vivo one does not expect the material of adverse reaction.

" biodegradable ": " biodegradable " used herein polymer is the polymer of (that is: being degraded to monomeric species) of can degrading fully under the condition in physiology or body.In preferred embodiment, described polymer and polymer biological degradation by-products are biocompatible.Biodegradable polymer needn't be for hydrolyzable degraded, and can need enzymatic catalysis to degrade fully.

" growth factor ": " growth factor " used herein is the chemicals of adjustable ganglion cell's metabolic process, and described cellular process includes but not limited to break up, synthetic and other metabolic activity of propagation, various cellular products.Growth factor can comprise a plurality of chemicals family, includes but not limited to cell factor, eicosanoid and differentiation factor.

" polynucleotides ", " nucleic acid " or " oligonucleotides ": term " polynucleotides ", " nucleic acid " or " oligonucleotides " refer to nucleotide polymer.Term " polynucleotides ", " nucleic acid " and " oligonucleotides " are used interchangeably.Usually, polynucleotides comprise at least three nucleotide.DNA and RNA all are polynucleotides.Described polymer can comprise natural nucleus glycoside (that is: adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, NSC 22837 and deoxycytidine), nucleoside analog (for example, 2-aminoadenine nucleosides, 2-thio-thymine nucleosides, inosine, pyrrolopyrimidine, 3-methyl adenine nucleosides, C5-propinyl cytidine, C5-propinyl uridine, C5-bromouracil nucleosides, the C5-fluorouracil nucleoside, C5-iodouracil nucleosides, C5-methylcystein nucleosides, 7-denitrogenation adenosine, 7-deazaguanine nucleosides, 8-oxo adenosine, 8-oxo guanosine, O (6)-methyl guanosine and 2-sulfo-cytidine), the chemical modification base, the bio-modification base (for example, methylated base), embed base, modification sugar (for example: 2 '-fluorine ribose, ribose, 2 '-deoxyribose, arabinose and hexose), or modify phosphate group (for example, thiophosphate and 5 '-N-phosphoramidite key).

" polypeptide ", " peptide " or " protein ": according to the present invention, " polypeptide ", " peptide " or " protein " comprise a chain that is connected together by peptide bond by three amino acid at least.Term " polypeptide ", " peptide " and " protein " are used interchangeably.Peptide can refer to an independent peptide or a peptide set.Although (that is: occurring in nature does not exist but can include compound in the polypeptide chain in can to select to use alpha-non-natural amino acid known in this technology; For example referring to http: ∥ www.cco.caltech.edu/ ^-Dadgrp/Unnatstruct.gif, it shows the structure of successfully including the alpha-non-natural amino acid in the functional ionic passage in) and/or amino acid analogue, but preferably, peptide of the present invention only comprises natural amino acid.Simultaneously, can be modified one or more amino acid in the peptide of the present invention, for example, by adding a chemical entities (such as a carbohydrate group, a phosphate groups, a farnesyl-group, a different farnesyl-group, a fatty acid group, a conjugation connector), functionalized or other modification, or the like.In a preferred embodiment, can produce a stabilized peptide (for example, having the longer half life period) more in vivo to the modification of peptide.These modifications can comprise amino acid whose the including in of cyclisation, D-of peptide, or the like.The peptide biological activity of expectation all should not significantly be disturbed in any modification.

" polysaccharide ", " carbohydrate " or " oligosaccharides ": term " polysaccharide ", " carbohydrate " or " oligosaccharides " refer to glycopolymers.Term " polysaccharide ", " carbohydrate " and " oligosaccharides " are used interchangeably.Usually, polysaccharide comprises at least three sugar.Described polymer can comprise natural sugar (for example, glucose, fructose, galactose, mannose, arabinose, ribose and wood sugar) and/or modify sugar (for example, 2 '-fluorine ribose, 2 '-deoxyribose and hexose).

" little molecule ": term used herein " little molecule " is used in reference to no matter be natural existence or the artificial molecule with a lower molecular weight of making (for example, by chemosynthesis).Usually, little molecule is monomer molecule and has a molecular weight less than about 1500g/mol.Preferable little molecule biologically active so that its can be in animal, preferably in mammal, more preferably in the mankind, produce a part or systemic effect.In some preferred embodiment, described little molecule is a medicine.Preferably, although and unnecessary, described medicine is for be considered as by relevant agency of government or department can be safely and the effective medicine of use.For example, the veterinary drug (it is incorporated herein with way of reference) that FDA lists for 500 to 589 times with medicine, at 21C.F.R. § § 21C.F.R. § § 330.5,331 to 361 and the mankind that list for 440 to 460 times is all thought can the medicine accepted used according to the invention.

" bioactivator ": " bioactivator " used herein is used in reference to the compound or the entity that can change, suppress, activate or otherwise influence the biological or chemical incident.For example, bioactivator can include but not limited to the anti-AIDS material, cancer-resisting substance, antibiotic, immunodepressant, antiviral substance, enzyme inhibitor, neurotoxin, opioid, hypnotic, anti-histamine substance, lubricant, sedative, anticonvulsant, muscle relaxant and Kang Pajinsenshi disease material, comprise channel blocker, myotic and anti-acetylcholine medicine are in interior antispastic and contraction of muscle agent, the anti-glaucoma compound, anti parasitic and/or antiprotozoan compound, cell-extracellular matrix interaction the conditioning agent that comprises cytostatic agent and anti-adhesive molecule, vasodilator, DNA, RNA or protein synthesis inhibitor, drug for hypertension, antalgesic, antipyretic, steroids and non-steroid anti-inflammatory agent, anti-angiogenesis, antisecretory factor, anticoagulant and/or antithrombotic agent, local anesthetic, medicament for the eyes, prostaglandin, antidepressants, the antipsychotic material, antemetic and developer.In certain embodiments, described bioactivator is a medicine.

One is applicable to " Pharmaceutical Substances:Syntheses; Patents, the Applications " of that the more complete list of bioactivator of the present invention and certain drug can be published by Thieme Medical Publishing referring to 1999, Axel Kleemann and Jurgen Engel; That edit by people such as Susan Budavari, as to publish by CRC publishing house " Merck Index:AnEncyclopedia of Chemicals, Drugs, and Biologicals " in 1996; The the UnitedStates Pharmacopei a-25/National Formulary-20 that calendar year 2001 is published by UnitedStates Pharmcopeial Convention company (Rockville MD), above-mentioned all publications all are incorporated herein with way of reference.

" tissue ": term used herein " tissue " refers to that one combines set and pericellular any extracellular matrix of the cell of carrying out a specific function by one or more.

Description of drawings

This paper is illustrated the present invention with reference to the some figures in the accompanying drawing, wherein:

Fig. 1 comprises through the antibody staining of its characteristic protein or through the light micrograph of histological stain with the control tissue of definite specificity and best dilution.(A and B) nestin, mouse embryo brain (embryo the 17th day); (C) β III-tubulin, mouse is subcutaneous; (D) cytokeratin-7, people's lung; (E) insulin, people's pancreas; (F) β III-tubulin, mouse brain; (G) vimentin, people's tonsil; (H) smooth muscle actin, people's tonsil; (I) CD34, people's tonsil; (J) CD31, people's tonsil; (K) albumin, liver; (L) alpha-fetoprotein (AFP), adult human liver; (M) sarranine-O, fibrocartilage.

Fig. 2 A comprises the light micrograph of differentiation hES cell (EB the 8th day), and described differentiation hES cell and matrigel mix to be incorporated in transforming growth factor (TGF), activin-A (ACT), retinoic acid (RA), insulin-like growth factor (IGF) or not have growth factor (CON) and have 2 weeks of growth down.Left figure: form the details in a play not acted out on stage, but told through dialogues image (ratio scale=1mm) of " spheroid ".Middle figure and right figure: use H﹠amp; The Histological section of the sample of E dyeing.Base map: formed " spheroid " cross section histochemistry and immunostaining in the matrigel that contains sarranine-O (saf0), anti-AFP and anti-nestin antibody (ratio scale=100 μ m).

Fig. 2 B to D shows the mechanical test result who has or do not have the PLGA/PLA supporting structure of matrigel.Wherein summarized the comparative result (D) of tensile strength test (B) and compression verification (C) and matrigel.

Fig. 3 is the gel photograph of a demonstration RT-PCR product, use ultra-high-sulfur(UHS) keratin (keratin), neurofilament heavy chain (NFH), cartilage matrix protein (CMP), alpha-fetoprotein (AFP), the primer of PDX-1 and GAPDH separate from 8 days embryoid (EB) RNA on carry out described RT-PCR, described embryoid is through trypsinization, through being seeded on the flat board by the fibronectin coating, and at transforming growth factor (TGF), activin-A (ACT), retinoic acid (RA), IDGF (insulin-like growth factor) (IGF), vascular endothelial growth factor (VEGF) exists down or 2 weeks of growth in the control medium (CON).

Fig. 4 comprise take from hEB (8 days), through control medium (CON) be supplemented with retinoic acid (RA) or the medium of IDGF (insulin-like growth factor) (IGF) is cultivated extra 2 weeks, and with the light micrograph (ratio scale=200 μ m) of the thick section of 5 μ m of the antibody staining of anti-human cell's keratin, alpha-fetoprotein and nestin.

Fig. 5 A to D is the scanning electron micrograph of PLLA/PLGA supporting structure, wherein (A) do not contain differentiation hES cell, (B to D) contains differentiation hES cell, among the figure with the adhesion condition (ratio scale: A, B=1mm, C=50 μ m, D=200 μ m) of different amplification showed cell to supporting structure.

Fig. 5 E to H comprises through haematine Yihong (H﹠amp; E) light micrograph of Ran Se PLLA/PLGA supporting structure.By with matrigel with cell inoculation (E, G) or by being coated with supporting structure (F, H) with fibronectin to supporting structure with hES cell inoculation (ratio scale=50 μ m) to supporting structure.

Fig. 5 I to K shows the propagation of cultivation two weeks back hES cells on the PLLA/PLGA supporting structure, wherein cultivate and with anti-BrdUrd antibody staining (brown) [(I) hanging down multiplication factor (100 times), (J to K) high-amplification-factor (1000 times)] (ratio scale=50 μ m) with BrdUrd.

Fig. 6 comprises not differentiation (undiff) or differentiation hES cell [embryoid (EB) 8 days] microphoto (diff), and described hES cell mixes with matrigel, is seeded on the PLLA/PLGA supporting structure, cultivates for two weeks, and uses H﹠amp; E or with the antibody staining (except that indicating is 400 times, and initial multiplication factor is 200 times) of anti-human alpha-fetoprotein (AFP), nestin or β III-tubulin.

Fig. 7 A comprises the light micrograph of hES cell-scaffold structure structure, described be configured in the control medium (CON) or in the presence of insulin-like growth factor (IGF) or retinoic acid (RA) 2 weeks of growth, through section and with anti-cell keratin antibody (redness), anti-vimentin antibody (green) and the DAPI (blueness) that is used for nuclear staining dye (ratio scale=100 μ m).

Fig. 7 B comprises the light micrograph of hES cell-scaffold structure structure, described be configured in the control medium (CON) or in the presence of transforming growth factor-beta (TGF β) or retinoic acid (RA) 2 weeks of growth, through section and with the agent of collagen trichrome stain dye (blueness) (ratio scale=100 μ m).

Fig. 7 C is the comparison diagram that is lined with the positive epithelial envelope cystic structures lumen diameter of cytokeratin in the structure in 2 weeks of growth in control medium or in the presence of IGF or RA.

Fig. 7 D be one show to use the positive staining area percentage that the anti-cell keratin antibody dyes (positive staining percentage, it obtains) in respectively carrying out two different experiments of twice in taking from the histologic section of sample and in normal human subject lung tissue (epithelial cell) section the positive staining area percentage (positive staining percentage) of dyeing figure (bar shaped represent mean value+/-SD).

Fig. 8 A shows the immunostaining of the histologic section of taking from the hES structure, describedly be configured in the control medium (CON) or in the medium of the combination (TGF/ACT) that is supplemented with TGF-β (TGF), activin-A (ACT), retinoic acid (RA), insulin-like growth factor (IGF) or TGF-β and activin-A, cultivated for 2 weeks, and with safranin O (Saf0) or with the antibody staining (ratio scale=50 μ m) that resists human AFP, albumin, nestin, β III-tubulin and S-100.

Fig. 8 B be a figure that is illustrated in the histologic section of taking from sample the positive staining area percentage that uses described coloring agent or antibody to dye (positive staining percentage, it obtains) in respectively carrying out three different experiments of twice (bar shaped represent mean value+/-SD).

Fig. 9 A is a demonstration RT-PCR result a gel photograph, described RT-PCR uses the primer of ultra-high-sulfur(UHS) keratin (keratin), neurofilament heavy chain (NFH), cartilage matrix protein (CMP), alpha-fetoprotein (AFP), PDX-1, CD34 and GAPDH to carry out on the RNA that separates the self-organizing structure, and described histological structure grew for 2 weeks in the presence of TGF-β (TGF), activin-A (ACT), RA, IGF or in the control medium (CON).

Fig. 9 B be one based on gene expression semi-quantitative analysis, various growth factor to the schematic diagram of the effect of tissue specific gene expression in the 3D structure (+=low the expression; ++ ++=high expressed).

Figure 10 A is a series of light micrographs of differentiation hES cell (EB the 8th day), wherein use matrigel with described hES cell inoculation (s+m) or after fibronectin coating, inoculate (s+fn) to the PLLA/PLGA supporting structure at supporting structure, in control medium (CON) or be supplemented with in the medium of TGF-β (TGF), activin-A (ACT), RA or IGF and cultivate, and after cultivating for two weeks, carry out immunostaining (ratio scale=50 μ m) through fixing, section and with the antibody of anti-CD31, anti-CD34 or anti-smooth muscle actin (SMA).

Figure 10 B is the figure [mean value of the different samples sections of 5 of numerical responses (± SD)] of the positive staining percentage (area of antibody positive cell in the histologic section) in the structure described in the exploded view 10A.

Figure 11 comprises the light micrograph of two all hES-supporting structure structures, and described hES-supporting structure structure is transplanted in the SCID mouse and used H﹠amp; E or with the antibody staining (ratio scale=50 μ m) of anti-human CD31, cytokeratin, AFP or β III-tubulin.

Figure 12 A comprises the light micrograph of the sample section (after two weeks) of PLLA/PGLA supporting structure, wherein inoculate described PLLA/PGLA supporting structure with differentiation human embryonic stem (hES) cell [embryoid (EB) the 8th day] and matrigel, and with the antibody staining that resists human desmin, myogenin and insulin.In described structure, find to finish protein positive cells, and some tanycytes.In described structure, do not find the myogenin cell.Seldom see the insulin positive cell.

Figure 12 B comprise through subcutaneous transplantation to the back of the body district of serious composite immune defective (SCI D) mouse and after 14 days in vivo with anti-Tra 1-60 and SSEA-4 antibody staining two week structure microphotos, contrast as one with being seeded to the supporting structure not differentiation hES cell of last 1 day (ES the 1st day).

Embodiment

In one embodiment, the present invention is a kind of method of generation one organizational project structure.The hES cell colony is exposed to one can promote one the expection differentiation pathway agent before or after, it is seeded on the carrier matrix.Described carrier matrix should have a sufficiently high modulus to resist collapse under the convergent force that applies at described cell.

We are surprised to find that, can set up a support environment and become three-dimensional (3D) institutional framework with guiding hES cell differentiation and tissue by making up suitable chemistry and physics inducement.We have used matrigel and biodegradable supporting structure to set up a series of 3D condition of culture, and find, the physics inducement that provides by biodegradable supporting structure can promote to organize the formation of spline structure.Specifically, being designed for the polymer scaffold that opposing is shunk under the compression stress that described cell applies can promote hES cell proliferation, differentiation and tissue to become the 3D structure.And the variation of growth factor condition can be induced the formation of the human tissue spline structure that comprises cartilage, liver and nerve fiber.At last, the hES cell that is incubated on the polymer scaffold can be organized into an endothelium pipe network structure in vitro, makes tissue blood vesselization.Therefore, become the 3D organizational aspects in the hES cell differentiation, physical environment is an influential parameter.

Can be when not having LIF and bFGF cultured cell with the formation of inducing embryoid body, and with after trypsinization.Can directly cell inoculation be made up to three dimensional matrix or with a gel that is used to inoculate.One exemplary gel is for reducing matrigel (the Growth-Factor Reduced Matrigel of growth factor ^TM) (matrigel), can buy from Becton-Dickinson.Not modified matrigel is the soluble basement membrane matrix (Kleinman, people such as H.K., Biochem.25:312,1986) of a kind of extraction from the EHS mouse tumor.The main component of this matrix is laminin, collagen I, nestin and Heparan sulfate proteoglycan (perlecan) (Vukicevic, people such as S., Exp.Cell.Res.202:1,1992).Produce matrigel (the Growth-FactorReduced Matrigel that reduces growth factor by from matrix, removing most of growth factor ^TM) (referring to people such as Taub, Proc.Natl.Acad.Sci.USA, (1990); 87 (10): 4002-6).Perhaps, described gel can be a collagen I gel.Spendable other gel of the present invention includes but not limited to alginates, fibrin, agar and collagen iv.

If use a gel, it also can comprise other extracellular matrix component, such as glucosaminoglycan, fibrin, fibronectin, proteoglycan and glycoprotein.Described gel also can comprise the basilar memebrane component, such as collagen iv and laminin.In one embodiment, the extracellular matrix component that is found in the tissue can be included in the gel, described tissue comprises the identical cell of cell type that is divided into stem cell.Can in this gel, add, also can add such as cell response conditioning agents such as growth factor and chemotactic factor (CF)s such as enzymes such as protease and clostridiopetidase As.

Gel will be absorbed on the matrix surfaces externally and internally, and can fill some micropores of a porous matrix.Before hardening, capillary force will make gel be retained on the matrix, perhaps allow gel hardening on matrix to have more from supporting to become.

Preferably, to such an extent as to described three dimensional matrix enough hard its can collapse under the convergent force that noble cells applies.As calculated, for the fibroblast that does not rely on supporting structure hardness, the average asymptotic power (Fcell) of each cell is about 3nN38.Although this is an extensively supposition,, will adopt following formula so if use described numerical value to represent an average cell applied force (σ):

In fact, available cross-sectional area (cell area) is estimated cell number in the cross section divided by single cell cross-sectional area (Acell).Above-mentioned equation can be expressed as following formula again:

$\mathrm{\σ}=\frac{F_{\mathrm{cell}}}{A_{\mathrm{cell}}}$

If the diameter of a cell is about 6 μ m in the supposition cross section, so, Acell is about 28 μ m (being assumed to a circular transverse cross-sectional).With the above-mentioned equation of these known numeric value substitutions, obtain following result: cell can apply the stress of an about 110Pa on a supporting structure.This is one quite general, estimated value widely.

In one embodiment, after being seeded on the matrix, embryonic stem cell can be kept three-dimensional structure, and, being reduced to of matrix cross-sectional area mostly is 50%, and for example, the matrix cross-sectional area is less than 40% with respect to a reduction without inoculation matrix, cell can be carried out various cell functions (for example, metabolic function, propagation, differentiation) simultaneously.In certain embodiments, under the mechanical force that institute's inoculating cell applies, the reduction of cross-sectional area be less than 30% or below, for example, be less than 20%, be less than 10% or be less than 1%.How the person of ordinary skill in the field can understand selective polymer and regulate its modulus (for example, by control molecular weight and crosslink density) so that the amount of contraction optimization.

In certain embodiments, formed matrix can have one be similar to the microstructure of alternative extracellular matrix microstructure.The molecular weight of also adjustable integral basis matter, tacticity and crosslink density control the engineering properties of matrix and degradation rate (for degradable supporting structure) the two.Also can make the engineering properties of engineering properties optimization with simulation implantation position place tissue.The shape and the size of final implant should be suitable for implantation position and types of organization.Matrix can only maybe can provide a structure or mechanical function as a stem cell delivery vehicle.Matrix can form arbitrary shape, for example, and as particle, cavernous body, pipe, spheroid, chain, coiled strand, the netted structure of capillary, film, fiber, network structure or thin slice.

Can control the matrix pores rate by the known multiple technologies of person of ordinary skill in the field.According to enough spaces being provided for cell and determining minimum-value aperture and degree of porosity to filter the needs that see through matrix and then arrive cell for nutrient provides enough spaces.Maximum diameter of hole and porosity are subject to the ability that matrix is kept its inoculation back mechanical stability.Along with the increase of porosity, use have one more high-modulus polymer, add harder polymer and all can be used to increase the stability of matrix for cellular contraction as a copolymer or mixture or the crosslink density that increases polymer.

Can make matrix by in the known multiple technologies of person of ordinary skill in the field any one.Salt leaching, micropore form technology, Solid-Liquid Separation (being called freeze drying sometimes) and contrary commentaries on classics autofrettage and all can be used to produce porous matrix.Can use the fiber traction and weave (referring to, for example, people such as Vacanti, (1988) Journal of Pediatric Surgery 23:3-9) produces the matrix with better arranged polymeric silk.The person of ordinary skill in the field it should be understood that and can utilize the standard polymers treatment technology to make the polymer substrate with multiple porosity and microstructure.

Preferably, polymer substrate is biodegradable.Be used to implement suitable biodegradable polymer of the present invention and known by affiliated technical field, it comprise poly-(lactic acid) (PLA), poly-(glycolic) (PGA) with PLA-PGA copolymer (PLGA).Other biodegradable material comprises PLA, poly-(acid anhydrides), poly-(carboxylic acid), poly-(ortho esters), poly-(fumaric acid propyl ester), poly-(caprolactone), polyamide, polyaminoacid, polyacetals, biodegradable polybutylcyanoacrylate, biodegradable polyurethanes and polysaccharide.Also can use simultaneously not biodegradable polymer.Other not biodegradable but still biocompatible polymer comprises polypyrrole, many aniline, polythiophene, polystyrene, polyester, not biodegradable polyurethanes, polyureas, poly-(ethylene vinyl acetate), polypropylene, polymethacrylates, polyethylene, Merlon and gathers (ethylene oxide).The person of ordinary skill in the field it should be understood that this is the polymer tabulation that an exemplary rather than comprehensively is applicable to organizational project.

The copolymer of above-mentioned polymer, mixture and adduct also can be used in the invention process.In fact, copolymer is particularly useful for the machinery and the chemical property of optimization matrix.For example, one for stem cell have high-affinity polymer can with a more hard polymer combination, to produce the matrix with the required hardness of opposing collapse.For example, PLA can make up with poly-(caprolactone) or PLGA and form a mixture.The two comes optimization matrix hardness can to adjust in a selected polymer and the copolymer polymer ratio.

PLA and PLA/PGA copolymer are particularly useful for forming biodegradable matrix.The erosion of polyester matrix is relevant with polymer molecular weight and degree of crystallinity.Higher molecular weight (for example, weight average molecular weight be 90,000 or more than) can produce the polymer substrate that its structural intergrity can keep longer time section; And lower molecular weight (for example, weight average molecular weight is 30,000 or following) can cause the shorter matrix life-span.But molecular weight and degree of crystallinity is the hardness of impact polymer matrix also.The tacticity of polymer also can influence modulus.Poly-(L-lactic acid) is isotactic polymer (PLLA), can increase crystallinity of polymer and the modulus that comprises the mixture of poly-(L-lactic acid).But the person of ordinary skill in the field it should be understood that the molecular weight of the above-mentioned arbitrary polymer of optimization and degree of crystallinity and controls matrix hardness.Similarly, can adjust the polymer ratio that has altogether in polymers or the mixture and obtain an expectation hardness.

In an exemplary embodiment, a cell response conditioning agent (such as the growth factor or a chemotactic factor (CF)) can be added in the polymer substrate.This conditioning agent can be used for promoting embryonic stem cell to be divided into an expectation target cell.Perhaps or in addition, can select conditioning agent that cell is raised to matrix, perhaps promote or suppress to raise to the specific metabolic activity of the cell of matrix.The exemplary growth factor includes but not limited to activin-A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, TGF-β, hepatocyte growth factor, platelet derived growth factor, TGF-α, IGF-I and II, hemopoieticgrowth factor, HBGF, peptide growth factor, erythropoietin(EPO), it is white plain to be situated between, TNF, interferon, colony stimulating factor, fibroblast growth factor, nerve growth factor (NGF) and muscle morphogenetic factor (MMF).The employed particular growth factor should be suitable for desired cellular activity and differentiation pathway.The regulating action of one extended familys growth factor is known by the person of ordinary skill in the field.

Before being seeded on the polymer substrate, also embryonic stem cell can be cultivated with growth factor or other cell response conditioning agent.These cells with combination of polymers before will begin the differentiation.Perhaps, the different cell colonys that are exposed to different cell response conditioning agents can be seeded on the different piece of three-dimensional polymer supporting structure.

Also can before inoculation, be added into other bioactivator, biomolecule and little molecule in the polymer substrate or be added in the medium.For example, can will promote fibronectin, integration element or the oligonucleotides (for example RGD) of cell adhesion to be added in the polymer substrate.Chemotactic factor (CF) or antiphlogistic can be added into the behavior that influences cell in the grafting matrix surrounding tissue in the matrix.

The polymer substrate through cell inoculation that contains or do not contain gel can be implanted in arbitrary tissue, comprise connective tissue, muscular tissue, nerve fiber and organ-tissue.Technology of the present invention can be used for forming the tissue of ectoderm, mesoderm and entoderm origin.In a preferred embodiment, selection can promote the ES cell differentiation and promote the growth factor that a predefined type tissue forms.For example, add the extracellular matrix that TGF-β can induce formation to have the cartilaginous tissue feature to the hES cell that is seeded on the three dimensional matrix.Activin A and IGF the two all can induce the generation of ES cell to have the protein of growing the liver feature.The ectoderm structure that RA can induce the hES groups of cells to be made into to be similar to nerve fiber.The ES cellular exposure can be promoted the formation of collagen and other bones ECM albumen in bone morphogenetic protein, bone specificity colony stimulating factor and/or PDGF.

When cell differentiation, can produce chemotactic factor (CF), chemotactic factor (CF) can with raise from the cell of surrounding tissue to through transplant, in the matrix of cell inoculation.The stem cell of transplanting with structure also will move out of matrix.The migration of cell helps to transplant structure and is integrated in the surrounding tissue.To move out peripheral vessels and of endothelial cell in grafting matrix, forming vasculature, for noble cells provides nutrition.

Stem cell is with expressing gene and the lucky protein with target cell feature that produced before it breaks up fully.Therefore, before stem cell was divided into other cell in liver cell and the liver fully, the stem cell that is exposed to activin A or I GF can be expressed the liver specificity gene.In fact, being exposed in the cell colony of a specific cells conditioning agent one is not all differentiation in the same manner of all stem cells.For example, some cells that are exposed to activin A or IGF can be expressed neural label or endothelial marker thing.These cells can help to form a neural network structure and a vasculature that is used to grow hepatic tissue.

And the mechanical interaction between cell and its extracellular matrix can influence cell processes.For further promoting to use the external source mechanical force to simulate the mechanical force that applies by tissue as a cell response conditioning agent along expectation approach differentiation.For example, when blood flow during through artery and vein endothelial cell be exposed under the shearing force.Because muscle is anchored on the bone at its two ends at least, therefore, muscle is exposed under all even non-homogeneous tensile stress simultaneously.In proper motion, bone will be subjected to compression and bending stress.Organ-tissue is exposed under liquid static stress and other compression., mechanical force is put on the matrix of cell inoculation the generation that can influence exciting albumen by the inoculation stem cell in vitro, influence the microstructure of the extracellular matrix of the degree of metabolic activity in cells and type and cell generation successively.

Equally, can use electro photoluminescence to influence cell differentiation and metabolism.For example, bone has piezoelectricity, and muscle response shrinks and relaxes in the signal of telecommunication by nerve conduction.The ES cell generation that the simulation expectation is organized the in vitro electro photoluminescence of electrical activity to make and is inoculated on the three dimensional matrix has the tissue that electrical feature is organized in this expectation.

Can be at shape and the microstructure and the external source power that puts on institute's seeding polymerization thing of particular organization's optimization polymer substrate.For example, with a pulse mode one medium is cycled through once inoculation tubulose matrix (promptly, one circumference stress) simulates the eparterial power that puts on, or use described medium to apply a shear stress (people such as Niklason on the inboard cell lining at pipe, (1999) science284,489-93; People such as Kaushall, (2001) Nat.Med., 7,1035-1040).Can be arranged institutional framework to polymer chain in the matrix, or be formed the formation that pipe network structure promotes vasculature with simulate muscular, tendon or ligament.

Even before inoculation ES cell breaks up fully, these cells also can will oneself be organized into the three-dimensional structure with nearly all animal tissue feature after being exposed to a cell response conditioning agent.Be inoculated in can provide on the matrix that ought to answer in all one's life to the mechanical force that stem cell applies after, stem cell can break up under the condition that more is similar to physiological environment than bidimensional culture dish and grow.In fact, before the final differentiation of ES cell, the integration of implant in a tissue location can be carried out sooner or be more effective.

Example

Experimental program

Cell culture

HES cell (H9 pure lines) is grown in rejects medium (KnockOut Medium) (Gibco-BRL, Gaithersburg, MD) (the Cell Essential of the mouse embryo fibroblasts in, Boston, MA) on, described in document 5, described medium is a modified form Du Beikeshi improvement Ying Shi medium at the ES optimum cellization.For inducing the formation of EB,, and in culture dish, be suspended in the differential medium of no LIF and bFGF 5 with the IV Collagen Type VI enzyme disassociation hES cell colony of 1mg/ml.

The preparation of supporting structure

Supporting structure is by poly-(lactic acid-be total to-glycolic) (Boeringer Ingelheim Resomer 503H, Ingelheim, Germany, Mn 25,000) and poly-(L-lactic acid) (Polysciences, Warrington, PA, Mn-300,000) 50/50 mixture constitute.Described in document 15, cavernous body is by the preparation of salt leaching method.When carrying out the cell differentiation experiment, with the rectangular block of cavernous body cutting into about 5 * 4 * 1mm3.Before cell inoculation, the sterilization of in 70% (vol/vol) ethanol, spending the night of these cavernous bodies, and washing 3 times in PBS.

Mechanical test

When only cavernous body being carried out extension test, dried cavernous body is trimmed to 0.4mm * 5mm * 11mm, and uses an Instron 5542 testing equipments till can't testing again with the strain rate of 0.05mm/ second.Use described Instron 5542 equipment in a parallel-plate dynamometer, to carry out compression verification on the cavernous body to independent cavernous body and matrigel (Growth Factor Reduced Matrigel) with minimizing growth factor.Cavernous body is that diameter is that 17mm, thickness are the porous disc of 0.8mm.Before with the test of 0.1mm/mm/ strain rate second, at first use 5% strain sample once to be circulated in advance with identical strain rate.

Cell differentiation on matrigel and the supporting structure

When in matrigel, inoculating, 8 to 9 days EB are carried out trypsinization, and with 0.8 * 106 mixing with cells in 25 μ l contain 50% (vol/vol) medium and reduce growth factor matrigel (BDBiosciences, Bedford, MA) in.The EB culture media supplemented has following growth factor: TGF-β 1 (2ng/ml), activin-A (20ng/ml) and IGF-I (10ng/ml) (R﹠amp; D Systems, Minneapolis, MN) and RA (300ng/ml) (Sigma).Under 37 ℃, in a 6-hole culture dish, solidify described mixture, and subsequently with sterile razor blade separating mixture from culture dish.Add each EB medium 4ml respectively.When on supporting structure, inoculating, with 25 μ l contain 50% (vol/vol) reduce the matrigel (GrowthFactor-Reduced Matrigel) of growth factor and separately the mixture of EB medium with 0.8 * 106 cell inoculation to each supporting structure.After the inoculating cell, its separately in the medium in a 6-hole culture dish suspension structure.For some experiments, supporting structure was soaked 1 hour in 50 μ g/ml fibronectins (Sigma), and before cell inoculation (no matrigel) is in 25 μ l EB medium, washing with PBS.

Tissue treatment and immunohistochemical staining

In 10% neutral formalin buffer solution, histological structure fixed 6 hours, handle and be embedded in the paraffin through routine.The thick transverse section of 5-μ m is placed on the silanization slide to carry out immunohistochemical staining or with haematine Yihong (H ﹠amp; E), trichrome stain agent or sarranine 0 dyeing.According to manufacturer specification, use the general HRP-DAB kit of Biocare medical science (Biocare Medical Universal HRP-DAB kit) (Biocare Medical, Walnut Creek, CA) carry out immunohistochemical staining, be to recover epitope, thermal treatment in advance 20 minutes under 90 ℃ in Re Veal buffer solution (Biocare Medical).Main antibody is the antibody of the human following albumen of mouse anti: desmin (1:150), alpha-fetoprotein (1:2500), cytokeratin 7 (1:25), CD31 (1:20)), albumin (1:100), vimentin (1:50), S100 (1:100) (above-mentioned) all from Dako, anti-human β III-tubulin (Sigma, 1:500), nestin (Transduction Laboratories, San Diego, CA, 1:1000), CD34 (Labvision, Fremont, CA, 1:20), SSEA4 (Hybridoma Bank, University ofIowa., Ames, 1:4) (grant University of Sheffield, Sheffield by Peter Andrews with Tra 1-60, U.K., 1:10).The mankind and mouse tissue (Dak) are with comparing to determine antibody specificity (Fig. 1).For proliferation research, before fixing with 10 μ l 5 '-bromo-2 '-deoxyuridine (BrdUrd) (Sigma) cultivated medium 3 hours.Use mouse anti BrdUrd antibody (1:1000) that histologic section is dyeed.

Be lined with the comparison of the lumen diameter of the positive epithelial envelope cystic structures of cytokeratin

Structure to two weeks of growth in control medium or in the presence of IGF or RA is handled, and as mentioned above with the dyeing of anti-cell keratin antibody.Counting tube blister-shape structure is measured lumen diameter and grouping (major diameter group＞200 μ m, intermediate diameters group＞40 μ m, minor diameter group＜40 μ m, sealing and multilayer tube chamber).The result who accounts for the percentage registration sample of tube chamber sum in each sample with tube chamber in each group be mean value (± SD), these results respectively carry out obtaining in twice the different experiments at two.

Reverse transcription (RT)-pcr analysis

(Qiagen, Chatsworth CA) separate total RNA to use RNEasy trace quantity reagent kit (RNEasy Mini Kit).Use Qiagen one one step RT-PCR kit (Qiagen OneStep RT-PCR kit) and 10 RNase of unit inhibitor (Gibco) and 40ng RNA to carry out RT-PCR.Described in primer sequence, reaction condition and cycle-index such as the document 7,15.(Gaithersburg MA) goes up the separation amplified production for E-Gel, Invitrogen at 1.2% Ago-Gel that contains the pyridine of bromination second.For some gels that comprise the RNA that uses a GADPH primer amplification, the mean pixel intensity by measuring each band and the mean pixel intensity that measured intensity is normalized to the GADPH band carried out semi-quantitative analysis.

Transplanting to the SCID mouse

With the differentiation hES in two weeks of growth on the supporting structure in vitro through subcutaneous transplantation in the back of the body district of 4 week SCID mouse in age (CB.17.SCID, Taconic Farms).The acellular supporting structure of being transplanted is with comparing.Transplanted back 14 days, and reclaimed implant, under 4 ℃, fixedly spend the night, be embedded in the paraffin, and section is for histological examination in 10% formalin buffer.

The result

Independent matrigel can not provide enough supports for three-dimensional hES cell differentiation

Shown that before this but the supportint cell tissue forms 14, in 15 the matrigel, cultivate differentiation hES cell (EB the 8th day) in the presence of the medium of the representative growth factor fill-in with the known ES of inducing cell differentiation, described growth factor fill-in has: retinoic acid (RA), activin-A, transforming growth factor (TGF-β) and insulin-like growth factor (IGF).Beginning is earlier made a plate-like with cell-matrix glue mixture, but in suspension culture after two weeks, described malformation is one " spheroid " shape, and this shows that cell shrinks matrigel.Can form little coalesced spheres with activin-A or with the sample that RA (can use TGF-β in a way) handles, with IGF or not contain sample that the control medium of growth factor handles then big and condense less (Fig. 2 A).

The histological examination of the spheroid of cultivating in IGF or control medium discloses: occur being lined with epithelial tubulose or cystic structures once in a while.On the contrary, the sample of handling with TGF-β, activin-A or RA does not comprise any this class formation, and individual cells is littler, and only has less intact cell epimatrix to produce (Fig. 2 A) usually.Spheroid in the latter organizes shows degeneration, and the cells in sample viability of handling at activated element-A is minimum.Though when handling with IGF or control medium, matrigel can support some to have the tubulose of open tube chamber or the formation of cystic structures, but the change of cell degradation, distortion and spheroid size shows that all independent matrigel is not enough to support growth of hES cell and 3D tissue to form.

Supporting structure can provide tolerance hES the machinery support of cellular contraction

Using biodegradable supporting structure to set up a 3D support environment is used to make the hES cell differentiation to become and is organized into and organize spline structure.Use one 50% poly-(lactic acid-be total to-glycolic) (PLGA) to make supporting structure with 50% poly-(L-lactic acid) mixture (PLLA).Selected PLGA (about 3 weeks) capable of being fast degraded inwardly grows to promote cell, and selected PLLA can provide mechanical hardness with opposing cellular contraction power.Select 250-500 μ m micropore size to promote cell inoculation and inwardly growth.

For determining whether supporting structure can tolerate the mechanical force that is applied by cell, and we compress and extension test.Carrying out compression verification on the independent PLLA/PLGA supporting structure with on the supporting structure with the matrigel (GrowthFactor-Reduced Matrigel) that reduces growth factor, and the result is being summarized among Fig. 2 B to C.Openly numerical value with these data and independent matrigel compares (Fig. 2 D) 16 then.Tensile property that supporting structure shows and consistent (Fig. 2 B, the D) 17 of the previous report numerical value of HMW PLLA supporting structure.Aspect compression, polymer scaffold has the modulus of compressibility of an about 65kPa.As by using the ANOVA statistical analysis determined, add matrigel and can not change modulus of compressibility (Fig. 2 C, D).Table of induction (Fig. 2 D) shows that the modulus of compressibility that supporting structure and matrigel/supporting structure showed is than high 3 orders of magnitude of modulus of compressibility of independent matrigel.This species diversity can influence the performance of the supporting structure with cell.Under the estimation cell compression of a 110Pa, supporting structure will shrink 0.2%, mean that it can resist contraction in fact.

Supporting structure supports the growth of hES cell attachment, differentiation and 3D tissue to form

For determining whether supporting structure works to hES cell differentiation and the formation of 3D tissue, we to through the differentiation hES cell in two weeks of growth on the culture dish of fibronectin coating with comparing on the supporting structure of fibronectin coating, cultivating the differentiation hES cell in two weeks, and differentiation in the independent matrigel and the differentiation that has in the supporting structure matrigel are compared.Bidimensional can be supported part cell differentiation (Fig. 3) through the culture dish of fibronectin coating, but can not support the 3D structure to form.Matrigel can form a 3D environment separately, but it can not support growth of hES cell and 3D tissue to form (Fig. 2).A kind of possibility is, matrigel and the difference that has between the supporting structure matrigel may partly be by due to the mechanical hardness of supporting structure separately, and the mechanical hardness of supporting structure is essential for opposing cellular contraction power.

When the differentiation of the structure of relatively cultivating through supporting structure and tissue form when forming with the differentiation of EBs and tissue, we find, more high expressed is arranged, this relevant with the epithelium tubular structure that more tissues form definition (Fig. 4) such as differentiation associated protein such as cytokeratin, AFP and nestins on supporting structure with nerve channel sample rosette.About the generation of extracellular matrix, in the EB that regulates through TGF-β, do not observe sarranine-O dyeing.EB colony is suitable heterogeneity on structure and protein expression level.Therefore, polymer scaffold shows to such an extent that be more suitable in promoting cell differentiation and homogeneous than EBs.

The two all can promote matrigel (Fig. 5 E, G) and fibronectin (Fig. 5 F, H) to break up hES (EB the 8th day) cell and anchor on the supporting structure, promote growth and cell viability.As the conventional organization of the histologic section got by SEM (Fig. 5 A to D) with different depth learn check shown in (Fig. 5 E to H), cell attachment is filled micropore in the whole surfaces externally and internally of supporting structure.Behind two time-of-weeks, the structure of cultivating with BrdUrd shows high level propagation and the viability (Fig. 5 I to K) that spreads all over supporting structure.Based on following observation, use differentiation hES cell to replace not breaking up the hES cell: to show obvious space with the supporting structure outer surface that does not break up the hES cell inoculation, and, (EB the 8th day) shows lower homogeneous growth and viability (Fig. 6 is also referring to Figure 12 A) in supporting structure central authorities when comparing with differentiation hES cell.

After the nurturing period, sample sets is made into the 3D form that is similar to institutional framework.For estimating these structures, the formation that we have analyzed epithelium and mesenchyma structure and extracellular matrix forms (Fig. 7) with tissue.When comparing with the formed envelope cystic structures of the control medium of no growth factor fill-in (65% ± 4＞40 μ m) (P＜0.01), add IGF can cause forming the cube that is lined with the cytokeratin positive to columnar epithelial cell than bassoon blister-shape structure (84% ± 6＞40 μ m, 10% ± 3＞200 μ m).On the contrary, RA can induce the formation of the structure with tube chamber, and described tube chamber is less than the tube chamber (25% ± 12＞40 μ m) (P＜0.01) of structure that control sample forms, and often produces ring-type multilayer or obturator (Fig. 7 A, C).RA handles and causes organizing the positive area percent of total of inner cell keratin to increase about 4 times (P＜0.01), approaches the level of finding in tested person adult epithelial tissue (Fig. 7 D).Shown in the trichrome stain of collagen (Fig. 7 B), cell structure is secretory cell epimatrix component towards periphery.But the collagen in the interpolation growth factor appreciable impact matrix forms and the tissue of intercellular matrix forms (Fig. 7 B).Newly form, organize bad collagen to be a little less than slightly fibrous and the dyeing in the control medium.The collagen that adds TGF β and can induce maturation, has dense dyeing band in medium forms, and RA suppresses collagen and forms.Regardless of condition, and compare with the structure in the sample of independent matrigel equivalent process, it is bigger and break up better to result from envelope cystic structures in the culture systems of supporting structure support and extracellular matrix.

Use biodegradable polymer scaffold to make up 3D mesoderm, ectoderm and entoderm institutional framework

We have further studied chemical inducement and the physics inducement combines for the effect that promotes to be divided into specific mesoderm, ectoderm and entoderm flat organization structure.Based in EB model and cell monolayer to the research 6-8 of mouse and human ES cell differentiation, we select the known growth factor that is divided into specific germinal layer of inducing.

For inducing the mesoderm tissue to form, we cultivate combining of cell and TGF-β, activin-A or TGF-β and activin-A two weeks.In this medium, add the formation that TGF-β can induce the cartilaginous tissue that spreads all over whole structure, shown in the high-level sarranine-O dyeing of glucosaminoglycan (GAG), have the feature 18 (Fig. 8) of cartilage cell epimatrix.On the contrary, add the formation (Fig. 8) that to induce safranin O-animus such as other growth factors such as activin-A (even when adding simultaneously), IGF and RA with TGF-β.Extraction the analysis showed that from the RT-PCR of heteroid RNA, compares with other sample, with the expression higher (Fig. 9 A) of cartilage matrix protein (CMP) in the sample of TGF-β processing.Understand according to us, these results show first, can use differentiation hES cell to form the 3D chondroid tissue.

The two all can induce the formation with the structure of growing the liver biochemical character to add activin-A or IGF.With compare, activin-A can induce high-caliber alpha-fetoprotein and albumin in whole sample.IGF can induce high-caliber AFP and albumin (Fig. 8) in more delimited area in structure, and do not observe dyeing when adding RA.These results show that in the hES 3D structure that supporting structure is supported, activin-A and IGF can induce the entoderm differentiation, and induce the formation that has with the tissue of growing the consistent biochemical characteristic of liver.Gene expression analysis shows, than handling with other growth factor, pancreas gene PDX-1 higher level (Fig. 9 B) in tissue-structure of handling with activin-A, this further supports activin-A inducing the hES cell to be divided into effect in the entoderm derived tissues on polymer scaffold.

For the ectoderm structure, we add RA7,8,19 in the structure medium.Compare with other growth factor, the RA fill-in can cause being lined with the preferential growth (Fig. 7) of epithelial entity and tubular structure.And, showing that with neural label dyeing cell can be organized into single or big multilayer nerve channel sample rosette structure, described structure is positive for nestin and β III-tubulin.Most of area of no rosette feature is dyed also to nestin and β III-tubulin be positive (Fig. 8).Be looped around around some pipes with the cell of the S-100 label of other neuroderm cell (colloid and) dyeing, performance one is supported or the migration phenotype.Compare with other sample, the gene expression analysis of the sample of handling with RA shows high-caliber keratin and neurofilament RNA and quite low mesoderm and entoderm gene expression (Fig. 9).These results show, but the differentiation of the ectoderm of the hES of RA induced growth on polymer scaffold has the tendency that goes up the higher structure consistent with nerve fiber on the form of being formed on biochemistry.

The contextual analysis of organization that is formed in the independent matrigel shows that chemokines can not be induced as being seen differentiation on supporting structure.It is opposite with the rosette spline structure to form tubulose when having RA, and cell will be organized into tuftlet on the matrigel, and these tuftlets have quite low nestin and express (if there is).Not observing AFP in the matrigel sample that activated element-A handles expresses.In IGF and control sample, can observe part A FP dyeing.In the sample that TGF-β handles, do not observing the cartilage sarranine-O of GAG dye (Fig. 2) that derives.These results show that supporting structure can promote in vitro three-dimensional cartilage, liver and neural sample tissue to exert an influence in forming.

The in vitro vascularization of three-dimensional tissue's structure

Because blood vessel can promote the formation 20-22 of complex organization's structure, therefore, we have analyzed whether the hES cell can break up and be organized into blood vessel in the institutional framework on being formed at supporting structure.The dyeing of carrying out with anti-CD34 and anti-CD31 antibody shows that after supporting structure was cultivated two time-of-weeks, cell can be divided into endothelial cell, and can be organized into the capillary structure that spreads all over tissue.The 3D culture of cell can promote to form a large amount of 3D blood vessel network structures, described 3D blood vessel network structure can closely interact with surrounding tissue (Figure 10).Comparison shows that of supporting structure medium vessels formation that has or do not exist matrigel do not needed matrigel, and this is because the sample that is seeded on the supporting structure (no matrigel) of fibronectin coating can produce higher levels of endothelium differentiation and vascularization (Figure 10).What is interesting is that the sample of handling through RA neither forms blood vessel (immunostaining by CD34 and CD31 shows), also do not express CD34 or CD31 (Fig. 9,10) shown in the RNA analysis.In addition, also detect the smooth muscle like cell of elongation.These cells are organized in around some tube chambers in the tissue, but in the sample of handling with RA not so (Figure 10).These results show that the differentiation hES cell that grows on the polymer scaffold can break up and form vascularization complex organization structure.And, can control this in vitro vascularization method by in medium, adding growth factor with supporting structure physical guide.

Two all backs are to the in vivo evaluation of three-dimensional tissue's structure

For analyzing the treatment potentiality of hES derived polymers supporting structure structure, we will construct through surgical graft in the s.c. tissue of SCID mouse in two weeks.When reclaiming implant (transplanting back 14 days), construct interior cell survival and do not detect the infection sign.Implant is not embedded in wherein by loose fiber granulation connective tissue fully, and has host blood vessel to penetrate.The immunohistochemical staining that uses human specific C D31 antibody to carry out shows, spread all over described structure exist immunocompetence (structure, Figure 11, arrow) and non-immunocompetence (host, Figure 11, arrow) blood vessel the two.In addition, construct the blood vessel of deriving and contain erythrocyte in the chamber, show that structure-host blood vessel is identical.The immunostaining that carries out with cytokeratin, β III-tubulin and AFP antibody shows, transplant structure and can defining in the supporting structure zone continue to express these human proteins (Figure 11) in the structure.After transplanting, still exist the continuation differentiation and the tissue of structure to form (Figure 11), the influence that the specific cells keratin was handled before it was transplanted in some instances.

When be configured in vivo continue maturation after, the structure of regulating through RA show than those in vitro being seen neuromechanisms (perhaps in vitro or in vivo using control medium) bigger and organize better neuromechanism, described neuromechanism comprises: be lined with the tubular structure of the high columnar epithelial cell that is surrounded by long cilium specimen chamber periosteum cell, and have the melanin granule of enriching (at H﹠amp; Be brown/black in the E section; Confirm data not shown by potassium permanganate dyeing) rosette.With β III-tubulin antibody to the neuroderm structure in the implant and on every side the mouse peripheral nerve fiber (Figure 11, asterisk) in the connective tissue dye.The dyeing of carrying out with SSEA-4 and Tra 1-60 antibody shows that all cells all breaks up (Figure 12 B).

Discuss

The two is all very important for the formation of human tissue sample 3D structure for physical environment and suitable growth factor fill-in.We prove, use the hES cell that grows on the polymer scaffold to form in vitro and have and human tissue of growing cartilage, liver, nerve and corresponding to form of blood vessel and biochemical character.We find that supporting structure can promote the formation of differentiated tissue.The cell characteristics that uses the fibroblast convergent force to simulate on the supporting structure can estimate that cell stress is 110Pa.Under this stress, matrigel will shrink 700%, and supporting structure only shrinks 0.2%, and this means that supporting structure does not shrink basically.Yet, visual cell's type and deciding, cell can represent different convergent forces.And chemical environment also plays a role in the cell mechanical property.Show that growth factor can influence the mechanical property 23-26 of the cell that comprises stem cell.This can be interpreted as what (IGF, or control medium) matrigel under some growth factor conditions shrink less, but under other condition (activin-A, RA) overall collapse (Fig. 2).When cell grows on the supporting structure that is supplemented with the isometric growth factor, can induce and further be divided into multiple particular cell types (such as endothelial cell, nerve cell, liver cell, or the like), be organized into 3D institutional framework (such as blood vessel network structure, nerve channel spline structure or the like) (Fig. 8 to 10) simultaneously.These results show that (for example: the machinery support that is provided by supporting structure) the two all can influence the differentiation of ES cell to complex organization for chemistry and physics inducement.

The effect of growth factor can cause directly differentiation or by promoting or suppressing differentiation or carry out cell by the apoptosis of inducing particular cell types and select.For example, when cell inoculation was on supporting structure, RA handled the specific differentiation that can induce to epithelium and neural spline structure, and suppressed mesoderm and entoderm differentiation (Fig. 8 to 10).As immunostaining 27 by AFP and albumin (two kinds of major protein) with liver differentiating characteristic, 28 and by shown in the expression 29 of pancreas gene PDX-1, add activin-A and can induce significant entoderm differentiation (Fig. 8,9) to being grown in hES cell on the supporting structure.Known activin-A mainly is an entoderm factor 6,30, and shown that in hES cell monolayer system activin-A can mainly induce mesoderm (mainly being muscle) differentiation, and the entoderm (comprising AFP and albumin) or the ectoderm gene 7 of not expressing any tested person.Yet, have report to show that activin-A can induce entoderm differentiation 31-33.Time of application (EB the 8th day to the 5th day) or three dimensions can necessarily act on the performance on the hES cell differentiation at activin-A.Another explanation for activin difference between the effects between two kinds of systems may be because the tissue blood vessel of 3D structural support forms the fact of (in the condition that allows the mesoderm differentiation).Show that recently endothelial cell and first angiogenic (even before blood vessel performance function) can provide inducement signal, described signal grows very important 34,35 for liver and pancreas.Therefore, the formation of a blood vessel network structure on supporting structure can be supported the inducing action of activin-A to the entoderm differentiation.

These results show, by using early stage differentiation hES cell and further inducing the differentiation of described hES cell in a supportive 3D environment (such as the PLLA/PLGA polymer scaffold), can produce the labyrinth of feature in vitro with various directed embryonic tissues.In vivo the result shows, can keep at least surviving 2 weeks through the hES of supporting structure support structure, and described structure can be raised and coincide with the host blood vessel system, and the differentiated form of in vitro inducing in vivo remaining intact property maybe can continue differentiation.Human tissue growth in vitro is hopeful to be used to solve organ shortage and infectious diseases risk, and this is the significant challenge in the present transplantation medicine.Except that potential clinical practice, in vitro tissue forms and can be early stage human grow and organ provides an important tool of research.

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The person of ordinary skill in the field is by understanding the present invention disclosed herein specification or the invention implementation content can be understood other embodiments of the invention.Specification of the present invention and example only should be considered as exemplary, and actual range of the present invention and spiritual by claims appointment of enclosing.

Claims (70)

1, a kind of organizational project structure, it comprises:

Embryonic stem cell;

A kind of three-dimensional cell carrier matrix, wherein said matrix can be resisted the convergent force that is applied by described stem cell; With

At least a growth factor selects it to be scheduled to the cell lineage differentiation or to promote described stem cell to be divided into a particular cell types along one in order to promote described stem cell.

2, organizational project structure as claimed in claim 1, wherein said stem cell is a mammalian embryonic stem cell.

3, organizational project structure as claimed in claim 2, wherein said cell is the hESC.

4, organizational project structure as claimed in claim 1, wherein said cell carrier matrix comprise one poly-(lactic acid)-poly-(lactic acid-be total to-glycolic) mixture.

5, organizational project structure as claimed in claim 4, wherein said cell carrier matrix comprises one poly-(L-lactic acid) and gathers 50/50 mixture of (lactic acid-be total to-glycolic).

6, organizational project structure as claimed in claim 1, a cross-sectional area of wherein said matrix reduces 50% at the most under a convergent force that is applied by described embryonic stem cell.

7, organizational project structure as claimed in claim 6, a cross-sectional area of wherein said matrix reduces 40% at the most under a convergent force that is applied by described embryonic stem cell.

8, organizational project structure as claimed in claim 7, a cross-sectional area of wherein said matrix reduces 30% at the most under a convergent force that is applied by described embryonic stem cell.

9, organizational project structure as claimed in claim 8, a cross-sectional area of wherein said matrix reduces 20% at the most under a convergent force that is applied by described embryonic stem cell.

10, organizational project structure as claimed in claim 9, a cross-sectional area of wherein said matrix reduces 10% at the most under a convergent force that is applied by described embryonic stem cell.

11, organizational project structure as claimed in claim 10, a cross-sectional area of wherein said matrix reduces 1% at the most under a convergent force that is applied by described embryonic stem cell.

12, organizational project structure as claimed in claim 1, wherein said cell carrier matrix further comprises a coating, and described coating comprises a kind of reagent that promotes cell adhesion.

13, organizational project structure as claimed in claim 12, the wherein said reagent of cell adhesion that promotes is selected from fibronectin, integration element and the oligonucleotides that can promote cell adhesion.

14, organizational project structure as claimed in claim 1, wherein said cell carrier matrix is biodegradable or not biodegradable matrix.

15, organizational project structure as claimed in claim 14, wherein said cell carrier matrix is selected from PLA, PGA, PLGA, poly-(acid anhydrides), poly-(carboxylic acid), poly-(ortho esters), poly-(fumaric acid propyl ester), poly-(caprolactone), polyamide, polyaminoacid, polyacetals, biodegradable polybutylcyanoacrylate, biodegradable polyurethanes, polysaccharide, polypyrrole, many aniline, polythiophene, polystyrene, polyester, not biodegradable polyurethanes, polyureas, poly-(ethylene vinyl acetate), polypropylene, polymethacrylates, polyethylene, Merlon, poly-(ethylene oxide), the copolymer of above-mentioned arbitrary polymer, the adduct of above-mentioned arbitrary polymer and above-mentioned arbitrary polymer, copolymer and adduct and alternative mixture.

16, organizational project structure as claimed in claim 1, it further comprises one or more biomolecule that place described cell carrier matrix, little molecule or bioactivator.

17, organizational project structure as claimed in claim 1, it further comprises a kind of gel, and described gel coating is at the surfaces externally and internally of described cell carrier matrix.

18, organizational project structure as claimed in claim 17, wherein said gel is selected from the matrigel (Growth Factor Reduced MATRIGEL) of collagen gel, alginates, agar and minimizing growth factor.

19, organizational project structure as claimed in claim 18, wherein said gel further comprises one or more in laminin, fibrin, fibronectin, proteoglycan, glycoprotein, glucosaminoglycan, chemotactic factor (CF) or the growth factor.

20, organizational project structure as claimed in claim 1, wherein said growth factor is selected from cell factor, eicosanoid and differentiation factor.

21, organizational project structure as claimed in claim 20, wherein said growth factor are selected from activin-A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, TGF-β, hepatocyte growth factor, platelet derived growth factor, TGF-α, IGF-I and II, hemopoieticgrowth factor, HBGF, peptide growth factor, erythropoietin(EPO), the white element of Jie, TNF, interferon, colony stimulating factor, fibroblast growth factor, nerve growth factor (NGF) and muscle morphogenetic factor (MMF).

22, organizational project as claimed in claim 1 structure, wherein said cell carrier matrix have a shape that is selected from particle, pipe, cavernous body, spheroid, chain, coiled strand, capillary network, film, fiber, network structure and thin slice.

23, a kind of method of generation one organizational project structure, it comprises:

One embryonic stem cell colony is provided;

Described embryonic stem cell is seeded on the cell carrier matrix; With described embryonic stem cell is exposed at least a selected agent promoting described stem cell along a predetermined cell lineage differentiation or promote described stem cell to be divided into a particular cell types,

Wherein said exposing step can be carried out before or after inoculation step, or the two carries out simultaneously.

24, method as claimed in claim 23, wherein said embryonic stem cell is a mammalian embryonic stem cell.

25, method as claimed in claim 24, wherein said embryonic stem cell is the hESC.

26, method as claimed in claim 23, wherein said cell carrier matrix is three-dimensional.

27, method as claimed in claim 23, a cross-sectional area of wherein said matrix reduces 50% at the most under a convergent force that is applied by described embryonic stem cell.

28, method as claimed in claim 27, a cross-sectional area of wherein said matrix reduces 40% at the most under a convergent force that is applied by described embryonic stem cell.

29, method as claimed in claim 28, a cross-sectional area of wherein said matrix reduces 30% at the most under a convergent force that is applied by described embryonic stem cell.

30, method as claimed in claim 29, a cross-sectional area of wherein said matrix reduces 20% at the most under a convergent force that is applied by described embryonic stem cell.

31, method as claimed in claim 30, a cross-sectional area of wherein said matrix reduces 10% at the most under a convergent force that is applied by described embryonic stem cell.

32, method as claimed in claim 31, a cross-sectional area of wherein said matrix reduces 1% at the most under a convergent force that is applied by described embryonic stem cell.

33, method as claimed in claim 23, wherein said cell carrier matrix comprise one poly-(lactic acid)-poly-(lactic acid-be total to-glycolic) mixture.

34, method as claimed in claim 33, wherein said cell carrier matrix comprise one poly-(L-lactic acid) and gather 50/50 mixture of (lactic acid-be total to-glycolic).

35, method as claimed in claim 23, it further comprises with a kind of reagent of cell adhesion that promotes and is coated with described cell carrier matrix.

36, method as claimed in claim 35, the wherein said reagent of cell adhesion that promotes is selected from fibronectin, integration element and the oligonucleotides that can promote cell adhesion.

37, method as claimed in claim 23, wherein said cell carrier matrix are biodegradable or not biodegradable matrix.

38, method as claimed in claim 23, wherein said cell carrier matrix is selected from PLA, PGA, PLGA, poly-(acid anhydrides), poly-(carboxylic acid), poly-(ortho esters), poly-(fumaric acid propyl ester), poly-(caprolactone), polyamide, polyaminoacid, polyacetals, biodegradable polybutylcyanoacrylate, biodegradable polyurethanes, polysaccharide, polypyrrole, many aniline, polythiophene, polystyrene, polyester, not biodegradable polyurethanes, polyureas, poly-(ethylene vinyl acetate), polypropylene, polymethacrylates, polyethylene, Merlon, poly-(ethylene oxide), the copolymer of above-mentioned arbitrary polymer, the adduct of above-mentioned arbitrary polymer and above-mentioned arbitrary polymer, copolymer and adduct and alternative mixture.

39, method as claimed in claim 23, it further comprises to described cell carrier matrix and adds one or more biomolecule, little molecule and bioactivator.

40, method as claimed in claim 23, it further comprises described embryonic stem cell is placed a gel, wherein described embryonic stem cell is seeded to comprise on the described cell carrier matrix described gel is placed on the surfaces externally and internally of described cell carrier matrix.

41, method as claimed in claim 40, wherein said gel are selected from the matrigel (Growth Factor Reduced MATRIGEL) of collagen gel, alginates, agar and minimizing growth factor.

42, method as claimed in claim 41, wherein said gel further comprises one or more in laminin, fibrin, fibronectin, proteoglycan, glycoprotein, glucosaminoglycan, chemotactic factor (CF) and the growth factor.

43, method as claimed in claim 23 is wherein cultivated and is carried out in a serum free medium.

44, method as claimed in claim 23, wherein said agent are selected from a growth factor, a mechanical force, a voltage, a biological activating agent, a biological molecule and a little molecule.

45, method as claimed in claim 44, wherein said growth factor is selected from cell factor, eicosanoid and differentiation factor.

46, method as claimed in claim 45, wherein said growth factor are selected from activin-A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, TGF-β, hepatocyte growth factor, platelet derived growth factor, TGF-α, IGF-I and II, hemopoieticgrowth factor, HBGF, peptide growth factor, erythropoietin(EPO), the white element of Jie, TNF, interferon, colony stimulating factor, fibroblast growth factor, nerve growth factor (NGF) and muscle morphogenetic factor (MMF).

47, method as claimed in claim 44, wherein said mechanical force are selected from the combination of circumference stress, shear stress, liquid static stress, compression, tensile stress and above-mentioned power.

48, method as claimed in claim 23, wherein said cell carrier matrix have a shape that is selected from particle, pipe, cavernous body, spheroid, chain, coiled strand, capillary network, film, fiber, network structure and thin slice.

49, method as claimed in claim 23 wherein provides to be included in growth factor existence cultivation embryonic stem cell down.

50, method as claimed in claim 49 is wherein cultivated and is carried out in a serum free medium.

51, a kind of organizational project structure, it comprises:

Embryonic stem cell;

A kind of three-dimensional cell carrier matrix, it comprises poly-(L-lactic acid) and gathers 50/50 mixture of (lactic acid-be total to-glycolic); With

TGF-β。

52, a kind of organizational project structure, it comprises:

Embryonic stem cell;

A kind of three-dimensional cell carrier matrix, it comprises poly-(L-lactic acid) and gathers 50/50 mixture of (lactic acid-be total to-glycolic); With

A member among activin A, the IGF, and above-mentioned arbitrary combination.

53, a kind of organizational project structure, it comprises:

Embryonic stem cell;

A kind of three-dimensional cell carrier matrix, it comprises poly-(L-lactic acid) and gathers 50/50 mixture of (lactic acid-be total to-glycolic); With

Retinoic acid.

54, as claim 51,52 or 53 described organizational projects structure, wherein said cell carrier matrix further comprises fibronectin or reduces in the matrigel (MATRIGEL) of growth factor one or more.

55, a kind of method that promotes to organize formation, it comprises:

One embryonic stem cell colony is provided;

Described embryonic stem cell is seeded to one to be comprised on the cell carrier matrix of poly-(L-lactic acid) and 50/50 mixture of poly-(lactic acid-altogether-glycolic); And

Described embryonic stem cell is exposed to TGF-β,

Wherein said cell can produce cartilaginous tissue.

56, a kind of method that promotes to organize formation, it comprises:

One embryonic stem cell colony is provided;

Described embryonic stem cell is seeded to one to be comprised on the cell carrier matrix of poly-(L-lactic acid) and 50/50 mixture of poly-(lactic acid-altogether-glycolic); And

Described embryonic stem cell is exposed among activin A and the IGF one or more,

Wherein said cell can produce alpha-fetoprotein and albumin.

57, a kind of method that promotes to organize formation, it comprises:

One embryonic stem cell colony is provided;

Described embryonic stem cell is seeded to one to be comprised on the cell carrier matrix of poly-(L-lactic acid) and 50/50 mixture of poly-(lactic acid-altogether-glycolic); And

Described embryonic stem cell is exposed to retinoic acid,

Wherein said cell can form the nerve fiber structure.

58, as claim 55,56 or 57 described methods, wherein said cell carrier matrix further comprises fibronectin or reduces in the matrigel (Growth Factor Reduced MATRIGEL) of growth factor one or more.

59,, wherein expose and comprise described cell carrier matrix through inoculation was cultivated for two weeks in vitro, and described method further comprises described cell carrier matrix through inoculation is transplanted in an animal as claim 55,56 or 57 described methods.

60, a kind of method that promotes to organize formation, it comprises:

One embryonic stem cell colony is provided;

Described embryonic stem cell is seeded on the cell carrier matrix;

Described cell carrier matrix through inoculation is cultivated one period scheduled time in the presence of a growth factor; And

Transplant in an animal through the cultured cells carrier matrix described.

61, method as claimed in claim 60, wherein said cell carrier matrix is selected from PLA, PGA, PLGA, poly-(acid anhydrides), poly-(carboxylic acid), poly-(ortho esters), poly-(fumaric acid propyl ester), poly-(caprolactone), polyamide, polyaminoacid, polyacetals, biodegradable polybutylcyanoacrylate, biodegradable polyurethanes, polysaccharide, polypyrrole, many aniline, polythiophene, polystyrene, polyester, not biodegradable polyurethanes, polyureas, poly-(ethylene vinyl acetate), polypropylene, polymethacrylates, polyethylene, Merlon, poly-(ethylene oxide), the copolymer of above-mentioned arbitrary polymer, the adduct of above-mentioned arbitrary polymer and above-mentioned arbitrary polymer, copolymer and adduct and alternative mixture.

62, method as claimed in claim 60, wherein said three-dimensional cell carrier matrix comprise poly-(L-lactic acid) and gather 50/50 mixture of (lactic acid-be total to-glycolic).

63, method as claimed in claim 60, it further comprises with a kind of reagent of cell adhesion that promotes and is coated with described cell carrier matrix.

64, as the described method of claim 63, the wherein said reagent of cell adhesion that promotes is selected from fibronectin, integration element and the oligonucleotides that can promote cell adhesion.

65, method as claimed in claim 60, it further comprises described embryonic stem cell is placed a gel, wherein described embryonic stem cell is seeded to comprise on the described cell carrier matrix described gel is placed on the surfaces externally and internally of described cell carrier matrix.

66, as the described method of claim 65, wherein said gel is selected from the matrigel (Growth Factor Reduced MATRIGEL) of collagen gel, alginates, agar and minimizing growth factor.

67, as the described method of claim 65, wherein said gel further comprises one or more in laminin, fibrin, fibronectin, proteoglycan, glycoprotein, glucosaminoglycan, chemotactic factor (CF) and the growth factor.

68, method as claimed in claim 60, wherein said growth factor are selected from activin-A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, TGF-β, hepatocyte growth factor, platelet derived growth factor, TGF-α, IGF-I and II, hemopoieticgrowth factor, HBGF, peptide growth factor, erythropoietin(EPO), the white element of Jie, TNF, interferon, colony stimulating factor, fibroblast growth factor, nerve growth factor (NGF) and muscle morphogenetic factor (MMF).

69, method as claimed in claim 60, the wherein said scheduled time was two weeks.

70, method as claimed in claim 60 is wherein cultivated and is carried out in a serum free medium.

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