CN100475956C - Method of preparing cell for transplantation - Google Patents
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Abstract
The invention describs a method of producing cells for transplantation into myocardial tissue of a mammal. The method comprises the steps: (a) providing bone marrow stem cells that have not been immortalized; (b) passage culturing two times said bone marrow stem cells under conditions that maintain the characteristics of bone marrow stem cells; (c) culturing said bone marrow stem cells in cardiac specific media to differentiate into cardiomyogenic cells; and (d) collecting the cells of step (c) when about 80 to 90% of said cells are cardiomyogenic cells. The cardiomyogenic cells induced according to the method can be clinically applied more suitably than those induced using demethylating agent such as 5-azacytidine, and therefore, they provide induced bone marrow stem cells of a mammal into cardiomyocytes having high rates of cell incorporation and cell survival for treating a mammal (e.g., a human) diagnosed as having a disorder characterized by insufficient cardiac function.
Description
Technical field
The present invention relates to produce the method for the cell that is used to transplant the cardiac muscle of mammal tissue.More specifically, relate to a kind of like this method, promptly with the biology somatomedin handle Mammals marrow stem cell make it to break up become heart cell system and with the bone marrow stem cell extracorporeal treatment of differentiation one period with reach efficiently that cell mixes (incorporation) rate and cell survival rate, to transplant the inductive bone marrow stem cell then interior and estimate the situation of its change to the mammalian body of suffering from myocardial infarction.
Technical background
The ability that the mescenchymal stem cell that obtains from adult's marrow has the various kinds of cell of being divided into has been shown, has comprised adipocyte, osteocyte, chondrocyte, liver cell and myocardial cell.Based on this ability, bone marrow stem cell is suggested the source as Transplanted cells.
Myocardial infarction is a kind of in the world one of dead major reason that causes.Because the myocardium archeocyte (cardiomyogenic cell) in grownup's heart can not increase, therefore because the myocardial cell's that myocardial infarction causes death meeting produces damage at the position of the cell existence of health.
The nearest bone marrow stem cell of discovering has significant adaptive faculty (flexibility) after being transplanted to heart.The bone marrow stem cell of being transplanted to cardiac muscle has the ability that differentiation becomes myocardium archeocyte of changeing, and this makes bone marrow stem cell can be used for cardiac muscle regeneration.Although because their potential therapeutic value, bone marrow stem cell can not differentiate enough myocardium archeocytes and improve function significantly.Therefore, the research that bone marrow stem cell is divided into the myocardial cell is carried out.
There is report to point out that for example the U-18496 processing can be so that the bone marrow stem cell differentiation become the myocardial cell with the demethylation medicine.U-18496 can make that general silencer begins to express with karyomit(e) nucleotide sequence demethylation randomly.Can induce into various kinds of cell through the bone marrow stem cell that U-18496 is handled, for example myocardial cell's (MyoD positive), osteocyte (the osteocalcin positive), adipocyte (the PPAR positive) and myocardium archeocyte (the myocardium calcium protein I positive).After bone marrow stem cell is exposed to U-18496, c-ab1 and interleukin-6 transcribe quick increase, and the expression decreased of main substrate protein collagen protein I.Yet demethylation medicine such as U-18496 are safe though be proved for human body, can not induce differentiation to become a kind of cell type, myocardium archeocyte significantly.
Based on above reason, inventor of the present invention thinks that bone marrow stem cell was exposed to and helps changeing differentiation in certain coenocorrelation and become myocardial cell system, and manages to find inducing cell to be divided into suitable the biology somatomedin and the condition of a kind of cell type (for example myocardium archeocyte) before transplanting.Therefore, the inventor has invented a kind of special method, utilizes the biology somatomedin to handle and bone marrow stem cell directly induced become myocardial cell system.And the inventor has also invented a kind of method, makes it to reach cell incorporation efficiency and cell survival rate efficiently in extracorporeal treatment bone marrow stem cell for some time before injection.This method is provided, and confirmation inductive Bone Marrow Stem Cells Transplantation can be improved myocardial infarction in the cardiac muscle of the dog that suffers from myocardial infarction.
The korean patent application No.10-2003-0004565 that ANTEROGEN company limited has has simultaneously described a kind of method from bone marrow stem cell High-efficient Production cardiac muscle archeocyte.Compare with previous patent, the invention provides a kind of more special and improved method.
Summary of the invention
Technical problem
The purpose of this invention is to provide a kind of method, it utilizes biology growth factor-induced Mammals marrow stem cell to become the myocardial cell, and before transplanting, the inductive bone marrow stem cell is handled for some time, to obtain cell incorporation efficiency and cell survival rate efficiently, then the bone marrow stem cell that obtains is used for treating the Mammals (for example human) of disease with myocardial function defective.
Technical scheme
According to one aspect of the present invention, a kind of method of producing the cell of transplanting the cardiac muscle of mammal tissue is provided, may further comprise the steps:
A) provide not by the bone marrow stem cell of immortalization;
B) described bone marrow stem cell is gone down to posterity under the condition that can keep the bone marrow stem cell feature 2 times;
C) described bone marrow stem cell is cultivated in the specific heart substratum, making it differentiation becomes myocardium archeocyte; And
D) after becoming myocardium archeocyte, collects about 80-90% of the cell of step (c).
In the method for the invention, described bone marrow stem cell can be from wanting transplanted Mammals.In step (c), described bone marrow stem cell was preferably cultivated 1 hour to 21 days, more preferably cultivated 6 days.
Specific heart substratum in the step (c) contains bFGF (Prostatropin), BMP-2 (bone morphogenetic protein-2) and IGF-1 (insulin-like growth factor-i).Preferably, substratum contains bFGF, BMP-2 and IGF-1, and wherein the concentration of every kind of factor is 1～200ng/ml, also contains L-xitix-2-PO of 2～20% foetal calf serums, 1～1000 μ M
4, the LIF of 5～15ng/ml and the dexamethasone (dexamethasone) of 1～200nM.
According to method of the present invention, the bone marrow stem cell of cultivation is expressed a kind of myocardium archeocyte specific marker of suitable transplanting.Preferably do not express in the proteic step of MF-20 and collect them at cell expressing MEF2 albumen.
The cell that is used to transplant the cardiac muscle of mammal tissue according to production of the present invention is undertaken by following steps: become the method for myocardial cell system transplanting the pre-induction bone marrow stem cell; With the inductive cell cultures regular hour to obtain the method for cell incorporation efficiency and cell survival rate efficiently; With cell is transplanted in the myocardial infarction Mammals model and is estimated the method for its effect.
The method that becomes myocardial cell system at transplanting pre-induction bone marrow stem cell may further comprise the steps:
(1) provides not by the bone marrow stem cell of immortalization;
(2) described bone marrow stem cell is gone down to posterity cultivation to obtain the cell of enough numbers;
(3) described bone marrow stem cell is cultivated in containing the specific heart substratum of biology somatomedin, making it differentiation becomes myocardium archeocyte;
(4) the cytodifferentiation state of monitoring step (3); And
(5) after becoming myocardium archeocyte, collects about 60-90% of the cell of step (3).
In addition, the method for cell incorporation efficiency and cell survival rate may further comprise the steps to obtain efficiently with inductive cell cultures for some time:
(1) provides not by the bone marrow stem cell of immortalization;
(2) described bone marrow stem cell is gone down to posterity cultivation to obtain the cell of enough numbers;
(3) described bone marrow stem cell was cultivated 1 hour to 21 days in containing the specific heart substratum of biology somatomedin, making it differentiation becomes myocardium archeocyte;
(4) at the differentiation state of the cell of specified time monitoring step (3); And
(5) after becoming myocardium archeocyte, collects about 80-90% of the cell of step (3).
In addition, the method for cell being transplanted in the Mammals model of myocardial infarction and being estimated its effect may further comprise the steps:
(1) prepares a myocardial infarction Mammals model;
(2) from described Mammals, separate bone marrow stem cell;
(3) described bone marrow stem cell is gone down to posterity cultivation to obtain the cell of enough numbers;
(4) described bone marrow stem cell was cultivated 6 days in containing the specific heart substratum of biology somatomedin, making it differentiation becomes myocardium archeocyte;
(5) after becoming myocardium archeocyte, collects about 80-90% of the cell of step (4); And
(6) described myocardium archeocyte is transplanted to Mammals.
The inventor utilizes the biology somatomedin to handle Mammals marrow stem cell and makes it differentiation and become myocardial cell system, before injection with cell cultures for some time to obtain in the cardiac muscle of damage cell incorporation efficiency and cell survival rate efficiently.Then cultured cells is transplanted in the cardiac muscle of mammiferous infarct, then the evaluate differentiation state.According to method inductive of the present invention cardiac muscle archeocyte than with the demethylation medicine for example the myocardium archeocyte of U-18496 inductive have better adaptive faculty.
To describe the present invention in detail below.
Be used for the step of the cell of external transplanting cardiac muscle of mammal tissue in production, somatomedin can be used to induce bone marrow stem cell to become myocardial cell system in all stages.Especially, all biology somatomedins can be used to induce the formation heart cell in the fetal development stage.These somatomedins include but are not limited to bFGF (Prostatropin), BMP-2 (bone morphogenetic protein-2) and IGF-1 (insulin-like growth factor-i), TGF-β, Wnt-inhibitor material, activin, vitamin A acid, Erb-2, all FGF somatomedin members, all TGF somatomedin members and all IGF somatomedin members.
The cell of these transplanting can increase or differentiation fully actively.Bone marrow stem cell can be induced specifically becomes myocardial cell system, and these clones can be identified with the heart specific mark, be used then.These marks include but are not limited to Nkx2.5, GATA4, MEF2 (myosin enhancement factor) and SRF (serum response factor).The myocardium archeocyte of differentiation can be applied equally, and these cells can be identified by the cardiac differentiation mark of expressing.These marks include but are not limited to MHC (myoglobulin heavy chain), cTnI (cardiac troponin I), cTnT (cardiac troponin T), α-cardiac actin, α-actinine and MLC2 (myosin light chain).Bone marrow stem cell can be taken as the myocardial cell and use, and these cells have the feature of mutual exchange message.The phenotype of these cells can be estimated by the expression of rhythmicity contraction and heart mark of correlation.The method of identifying these heart mark of correlations includes but are not limited to any detectable method, and for example reporter gene is expressed (by specific heart promotor inductive LacZ), rna expression (RT-PCR, Northern trace, RNase guard method) or protein expression (immunofluorescence analysis, Western trace, flow cytometry).
The cell of transplanting according to aforesaid method can be to be in myocardium archeocyte any cell of differential period.
Be meant the cell-derived stem cell of medulla mesenchyma at " mesenchymal stem cells MSCs or bone marrow stem cell (BMSC) " mentioned in this article, it is CD45
-Mesenchymal stem cells MSCs also can be called bone marrow stem cell and marrow multipotency progenitor cell.
" heart cell " is meant a kind of heart cell (for example myocardial cell) of differentiation or a kind ofly will produces or break up the cell (for example cardioblast or myocardium archeocyte) that becomes heart cell.
" myocardial cell " is meant the muscle cell in a kind of heart, but it can express the cardiac marker (for example Cx43 in α-myoglobulin heavy chain, cTnI, MLC2v, α-cardiac actin and the body) of detection limit, can shrink still and can not breed.
" cardioblast " but be meant and a kind ofly can express the cardiac marker of detection limit and can shrink and proliferating cells.
" myocardium archeocyte " but but be meant a kind of Csx/Nkx2.5RNA that can express detection limit or albumen but do not show organized muscle segment structure or contraction and preferably do not express the proteic cell of myoglobulin heavy chain of detection limit.
" cardiac specific " is meant the cardiac differentiation initial period, and wherein initial heart transcription factor is prior to the expression of final differentiation marker and express.
" specific heart substratum " is meant the substratum of a kind of bFGF of comprising, BMP-2 and IGF-1, and it induces bone marrow stem cell to become the myocardial cell.
" commentaries on classics differentiation " is meant and is divided into and its other different cell type of germinal layer source.
Other features and advantages of the present invention will be tangible by the description of following preferred embodiment and claim.
Beneficial effect
Can use clinically according to inductive of the present invention cardiac muscle archeocyte, than with the demethylation medicine for example the myocardium archeocyte of U-18496 inductive have and be more suitable for.According to the present invention, providing becomes myocardial cell's inductive Mammals marrow stem cell, it has cell incorporation efficiency and cell survival rate efficiently, and described inductive bone marrow stem cell can be used for treating the Mammals (for example human) that suffers from the heart function deficiency disorders.
Description of drawings
Above-mentioned and other purpose of the present invention, feature and advantage are by following detailed description and can more be expressly understood in conjunction with the accompanying drawings, wherein:
Fig. 1 is for differing Photomicrograph (* 100) from isolating, the bone marrow stem cell cultivated of dog.
Fig. 2 is a series of Photomicrographs (* 100), shows the cellular form after the dog bone marrow stem cell is cultivated in the specific heart substratum and the expression of MEF2 (A) and desmin (B), and (C) is the negative control of MF-20.
Fig. 3 is a series of Photomicrographs (A is * 200 to F, and G is * 100 to N), shows the expression with heart specificity substratum treatments B MSC and dyeing back MEF2.A and D represent untreated cell; B and E show with the cell of heart specificity substratum processing after 1 day; C and the cell of F display process after 2 days; G and the cell of K display process after 3 days; H and the cell of L display process after 6 days; The negative contrast of I and M, it was handled 8 days and dyeed with rabbit-IgG.
Fig. 4 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 15 days.Red fluorescence is represented the BMSC that is transplanted to host's cardiac muscle of DiI mark, and green fluorescence represents that blue-fluorescence is represented the nucleus with the DAPI mark with the painted myocardium archeocyte of MF-20 antigen.
Fig. 5 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 70 days.Red fluorescence is represented the BMSC that is transplanted to host's cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with MHC, and blue-fluorescence is represented the nucleus with the DAPI mark.
Fig. 6 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 70 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with cTNI.
Fig. 7 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 96 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with MF-20.
Fig. 8 for show inductive BMSC be transplanted in the cardiac muscle of dog of infarct before (BL) and the figure of (4,8,12 week) changes in heart rate afterwards.
Fig. 9 is a series of Echocardiograms, show inductive BMSC be transplanted in the cardiac muscle of dog of infarct before (BL) and afterwards (4,8,12 week) shorten the variation of mark (fractionalshortening) and shortening area (area shortening).
Figure 10 is an Echocardiogram, show inductive BMSC be transplanted in the cardiac muscle of dog of infarct before (BL) and (4,8,12 week) variation of wall thickness afterwards afterwards.
Optimal mode
Hereinafter, will be described in detail the present invention in conjunction with a plurality of examples.These examples are usefulness as an illustration only, does not represent the present invention to be confined to these examples.Simultaneously, though the bone marrow stem cell in these examples is separated from dog, do not represent the present invention to be confined to this, the present invention can be used for separating from all mammiferous cells, comprises the mankind, rat, mouse, monkey or the like.
Embodiment 1: the method for inducing myocardium archeocyte by handling with the biology somatomedin from bone marrow stem cell
Produce myocardial infarction surpass around after, the syringe that soaked with heparin obtains the bone marrow stem cell of 15-20ml from the dog ilium.These bone marrow stem cells are containing 2～20% foetal calf serums, 1～1, L-xitix-2-PO of 000 μ M
4, the LIF (leukaemia inhibitory factor) of 5～15ng/ml and 1～200nM the substratum of dexamethasone in cultivate.Before accepting bone marrow stem cell, all culture apparatuses and slide all at room temperature use 5ng/ml collagen protein bag by 15 minutes.This conditions in vitro can be so that bone marrow stem cell be kept the feature of its self, and can not lose by going down to posterity amplification differentiation medicament replying of somatomedin for example.And this condition can also allow bone marrow stem cell cultivate maintenance mesenchyme form and caryogram by going down to posterity.Fig. 1 is for differing Photomicrograph (* 100) from isolating, the bone marrow stem cell cultivated of dog.
Go down to posterity cultivate twice after, bone marrow stem cell is inoculated into 4 holes and cultivates on the slide, and with myocardium division culture medium processing.Division culture medium is DMEM, comprising 2～20% foetal calf serums, 1～1, and L-xitix-2-PO of 000 μ M
4, 5～15ng/ml LIF, 1～200nM dexamethasone, 1～200ng/ml bFGF, 1～200ng/ml BMP2 and 1～200ng/mlIFG-1.Contain the known somatomedin that period heart development is played an important role in fetal development in the substratum.
After cultivating 14 days under the condition that somatomedin exists, about 50～90% bone marrow stem cell is expressed MEF2 albumen.Fig. 2 is a series of Photomicrographs (* 100), shows the cellular form after the dog bone marrow stem cell is cultivated in the specific heart substratum and the expression of MEF2 (A) and desmin (B), and (C) is the negative control of MF-20.
In these cells, find MEF2 albumen, yet the expression of cardiac differentiation mark does not find that but these expression can be by identifying with the immune response of MF-20.Therefore cell expressing specific heart mark shown in showing, and do not express last differentiation marker eventually.
It is specific mark that the expression of MEF2 can be used as a kind of heart cell, and expressed proteins is positioned at nucleus.Before the fetal development stage, cardiac differentiation was induced, the MEF2 transcription factor was expressed in the heart progenitor cell.These genes are expressed in the heart mesoderm before embryo's method etap early stage.In addition, MEF2 albumen is to express the important transcription factor that heart breaks up necessary many structure genes fully.
MEF2 or myoglobulin heavy chain be mark by the following method, makes it to be detected in cell: the cultured cells process is freezing, and fixes 20 minutes in 4% formaldehyde, handles 15 minutes with the PBS that contains 0.2%Triton X-100 then.After washing 3 times with PBS again, product was handled 15 minutes with trace solution (PBS contains 1% fetal bovine serum albumin and 0.2%Tween 20).Sample of handling and anti-MEF2 (1: 200, sc-10794, Santa Cruz Biotechnology, Santa Cruz), MF-20 is (1: 100, Developmental Studies Hybridoma B ank, University of Iowa, Iowa City, Iowa), resistive connection albumen (1: 100～200, Sigma-Aldrich) and if desired react in the box of preserving moisture with a kind of isotype antibody (the mouse IgG2b of rabbit igg 2b, the MF-20 of MEF2, the mouse IgG1 of desmin), 4 ℃ are spent the night.The sample slide is washed 3 times with washing lotion (PBS that contains 0.5%Tween 20), then according to the explanation of producer's handbook, with second antigen (the anti-rabbit igg of the donkey of MEF2, the anti-mouse IgG of the donkey of MF-20 and desmin; Molecular Probe) reaction.Through 3 cleanings, the sample slide is observed down at fluorescent microscope (NikonTS 100).
Embodiment 2: bone marrow stem cell is induced for some time to become to have the method for the myocardium archeocyte of cell incorporation efficiency and cell survival rate efficiently
Bone marrow stem cell can be handled the different time with the biology somatomedin in the specific heart substratum, from 1 hour to 21 days, during this period of time, about 40～90% cell expressing nucleoprotein MEF2.According to difference, induce intentionally the ratio of myogenous cells can bring up to 60～90% with the treatment time in the heart specificity substratum.Handle Best Times and can make 80～90% cell expressing MEF2 albumen.Transplant the proteic cell of these 80～90% expression MEF2, the heart of the infarct of can the most compatibly regenerating.
Cultivated 6 days the specific heart substratum from the isolated bone marrow stem cell of adult dog, under fluorescent microscope, be illustrated in and have the proteic optimum quantity of MEF2 in the nucleus.These cells are transplanted to these cells mix in the cardiac muscle shown in the dog of infarct, after transplanting, dog has survived 96 days.Fig. 7 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 96 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with MF-20.This in vivo test is carried out by the following method:
In order to determine best induction time, the dog bone marrow stem cell is through 2 cultivations of going down to posterity, and cultivate on the slide in 4 holes that cultured cells are inoculated into collagen protein bag quilt, and concentration is 10,000 cell/sheets.After 24 hours, cell washes twice with PBS, adds 1 milliliter of specific heart substratum.One of them slide does not add the specific heart substratum as negative control.The specific heart substratum was handled after 1,2,3,4,5,6,8 and 10 day, and fixed cell is also used the MEF2 antibody staining, observes under fluorescent microscope then.Fig. 3 is a series of Photomicrographs (A is * 200 to F, and G is * 100 to N), shows with heart specificity substratum treatments B MSC and the proteic expression of dyeing back MEF2.A and D represent untreated cell; B and E represent through 1 day cell of specific heart substratum processing; C and F represent to handle the cell after 2 days; G and K represent to handle the cell after 3 days; H and L represent to handle the cell after 6 days; The negative contrast of I and M is dyeed through 8 days processing and with rabbit igg.According to the painted relative intensity of MEF2 in the test-results, it is the highest to handle after 6 days expression amount through the specific heart substratum.
Based on The above results, be used for the in vivo test of the dog class model of myocardial infarction with the cell of heart specificity substratum processing collection after 6 days.
Embodiment 3: bone marrow is in cell and estimate cell incorporation efficiency and survival rate
Vitro differentiation intentionally the bone marrow stem cell of myogenous cells system be transplanted to the cardiac muscular tissue of the dog of infarct.The myocardial infarction of dog continues inaccessible the generation by arteria coroaria sinistra.Before the bone marrow stem cell, infarct kept stable 2 months at least.For fear of the immunological rejection of graft, following marrow and the preparation bone marrow stem cell from each transplant recipient dog, collected:
In about 4 weeks after the ligation, after making a definite diagnosis myocardial infarction with Echocardiogram, marrow is drawn in the perforation back on ilium.Rapidly marrow and heparin are mixed, freezing back is transported to tissue culture room with dry ice, there marrow is thawed at 37 ℃, rocks, and washes once with conventional DMEM, places and contains substratum (2～20% foetal calf serums, 1～1000 μ M L-xitix 2-PO
4, 5～15ng/ml LIF and 1～200nM dexamethasone) tissue culture flasks in.Go down to posterity cultivate twice after, bone marrow stem cell was with the culture medium culturing that contains 1～200ng/ml bFGF, 1～200ng/ml BMP2 and 1～200ng/ml IFG-1 6 days.When results, bone marrow stem cell is used for following the tracks of survival and the growing state of transplanting the back cell with a kind of red fluorescence mark DiI mark.Approximately gathered in the crops 2 * 10
8Individual cell is expelled to the infarcted region of heart.
Transplant the survival of back bone marrow stem cell and can use DiI Fluirescence observation judgement after death.Fig. 4 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 15 days.Red fluorescence is represented the BMSC that is transplanted to host's cardiac muscle of DiI mark, and green fluorescence represents that blue-fluorescence is represented the nucleus with the DAPI mark with the painted myocardium archeocyte of MF-20 antigen.As shown in Figure 4, transplant and observed a large amount of DiI positive cells at cardiac muscle in back 15 days, the long-term surviving of bone marrow stem cell is described.Especially, in the myocardial region that contains the MF-20 positive cardiomyocytes and lack the stem cell that observes the DiI mark in the infarcted region of MF-20 positive cardiomyocytes.
In order to study the long-term surviving of transplanted cells, described dog is condemned to death and observes.Fig. 5 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 70 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with MHC, and blue-fluorescence is represented the nucleus with the DAPI mark.Fig. 6 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 70 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with cTNI.
In addition, Fig. 7 is Photomicrograph (* 40), shows to transplant the transplanted position of BMSC in the cardiac muscle of the dog of infarct after 96 days.Red fluorescence is represented the BMSC that is transplanted to cardiac muscle of DiI mark, and green fluorescence is represented the antigen reactive myocardium archeocyte with MF-20.Shown in Fig. 5 to 7, the cell of DiI mark is all observed in the zone of infarct in all examples.In some example, DiI fluorescence is observed in the place of the specific heart mark that contains MF-20.In addition, contain the positive stem cell of DiI at the fringe region of infarcted region, it also expresses myocardium specific mark MHC α/β and TNI.
These data declarations can survive and mix in host's cardiac muscle and the signature of expressing cardiac differentiation at BMSC external conditioning as stated above, that transplant.
Embodiment 4: the improvement of myocardial infarction
Above-mentioned dog class myocardial infarction model is estimated the recovery effect that BMSC transplants by Echocardiogram (ECG).Carry out ECG after 3.5,4.5 and 5 weeks after BMSC transplants, and with transplant before ECG relatively.
Fig. 8 is a graphic representation, shows that inductive BMSC is transplanted to (BL) and the variation of (4,8 and 12 week) heart rate afterwards before the dog cardiac muscle of infarct.Fig. 9 is a series of Echocardiograms, show inductive BMSC be transplanted to (BL) before the dog cardiac muscle of infarct and afterwards (4,8 and 12 week) shorten the variation of mark and shortening area.In addition, Figure 10 is an Echocardiogram, shows that inductive BMSC is transplanted to (BL) and (4,8 and 12 week) variation of wall thickness afterwards afterwards before the dog cardiac muscle of infarct.
Shown in Fig. 8 to 10, in all animals, it is more synchronous with contiguous myocardial region that the contraction of infarcted region becomes.ECG result has confirmed that the histology among the embodiment 3 finds, and proof is transplanted the BMSC that cultivates and caused after the infarct part of heart tissue to be restored.
Claims (5)
1, a kind of production is used for being transplanted to the method for the cell of cardiac muscle of mammal tissue, comprises step:
(a) provide not by the bone marrow stem cell of immortalization;
(b) above-mentioned bone marrow stem cell is gone down to posterity under the condition that can keep the bone marrow stem cell feature cultivate 2 times;
(c) above-mentioned bone marrow stem cell is cultivated in the specific heart substratum, making it differentiation becomes myocardium archeocyte, and described specific heart substratum contains bFGF (Prostatropin), BMP-2 (bone morphogenetic protein-2) and IGF-1 (insulin-like growth factor-i) as the biology somatomedin; With
(d) after the about 80-90% of cell of step (c) becomes myocardium archeocyte, collect the cell of expressing MEF2 albumen and not expressing MF-20.
2, the process of claim 1 wherein that described bone marrow stem cell comes from the Mammals that will transplant.
3, the process of claim 1 wherein that described bone marrow stem cell cultivated 1 hour to 21 days in step (c).
4, the method for claim 3, wherein said bone marrow stem cell were cultivated 6 days in step (c).
5, the process of claim 1 wherein that described substratum contains bFGF, BMP-2 and IGF-1, wherein the concentration of every kind of factor arrives 200ng/ml 1, and also contains L-xitix-2-PO of 2～20% foetal calf serums, 1～1000 μ M
4, the LIF of 5～15ng/ml and the dexamethasone of 1～200nM.
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| KR1020030088850 | 2003-12-09 | ||
| KR1020030088850A KR100674783B1 (en) | 2003-12-09 | 2003-12-09 | Method of preparing cell for transplantation |
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| CN1890365A CN1890365A (en) | 2007-01-03 |
| CN100475956C true CN100475956C (en) | 2009-04-08 |
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| JP (1) | JP4122365B2 (en) |
| KR (1) | KR100674783B1 (en) |
| CN (1) | CN100475956C (en) |
| AU (1) | AU2004296369B2 (en) |
| WO (1) | WO2005056779A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474456A (en) * | 2016-10-18 | 2017-03-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cell preparation and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100694442B1 (en) | 2001-04-13 | 2007-03-12 | (주)안트로젠 | Encapsulated cell indicator system |
| US7534607B1 (en) | 2005-12-27 | 2009-05-19 | Industrial Technology Research Institute | Method of producing cardiomyocytes from mesenchymal stem cells |
| KR20070070445A (en) * | 2005-12-29 | 2007-07-04 | (주)안트로젠 | Method of preparing cell for heart tissue regeneration |
| US8153427B2 (en) | 2007-03-15 | 2012-04-10 | California Stem Cell, Inc. | Cardiomyocytes and methods of producing and purifying cardiomyocytes |
| US9446076B2 (en) * | 2009-05-20 | 2016-09-20 | Cardio3 Biosciences S.A. | Pharmaceutical composition for the treatment of heart diseases |
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| CA2395950A1 (en) * | 1999-12-28 | 2001-07-05 | Kyowa Hakko Kogyo Co., Ltd. | Cells capable of differentiating into heart muscle cells |
| WO2002083864A2 (en) * | 2001-04-13 | 2002-10-24 | Anterogen Co., Ltd. | Methods and reagents for cell transplantation |
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| CN106474456A (en) * | 2016-10-18 | 2017-03-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cell preparation and its preparation method and application |
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| Publication number | Publication date |
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| AU2004296369B2 (en) | 2008-05-01 |
| JP2007513625A (en) | 2007-05-31 |
| AU2004296369A1 (en) | 2005-06-23 |
| KR20050055823A (en) | 2005-06-14 |
| CN1890365A (en) | 2007-01-03 |
| JP4122365B2 (en) | 2008-07-23 |
| KR100674783B1 (en) | 2007-01-25 |
| WO2005056779A1 (en) | 2005-06-23 |
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