CN1536076A - Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells - Google Patents
Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells Download PDFInfo
- Publication number
- CN1536076A CN1536076A CNA03109287XA CN03109287A CN1536076A CN 1536076 A CN1536076 A CN 1536076A CN A03109287X A CNA03109287X A CN A03109287XA CN 03109287 A CN03109287 A CN 03109287A CN 1536076 A CN1536076 A CN 1536076A
- Authority
- CN
- China
- Prior art keywords
- cell
- mscs
- mesenchymal stem
- amplification
- myocardium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention is aimed at providing a method for mass-amplifying human bone marrow mesenchymal stem cell (MSCs) and making external directed induction and differentiation to obtain myocardium-like cell. In order to implement the above-mentioned aim said invention adopts the following steps: (1). utilizing biological property of MSCs and adopting density gradient centrifugation method to separate and enrich human MSCs, utilizing self-renewal and multiplication characteristics of stem cell to make amplification of stem cell; (2). making directed induction in vitro by using specific chemical induction agent; and (3). making external induction and differentiation to obtain the myocardium-like cell. Said also provides its application for curing diseases of heart.
Description
Technical field
The present invention relates to a kind of biological characteristics that utilizes mesenchymal stem cells MSCs, separate amplification adult bone bone marrow-drived mesenchymal stem, add the finite concentration chemical inducer, directional induction in vitro is divided into the method for cardiac-like muscle cell.
Background technology
Coronary heart disease myocardial infarction and heart failure are one of principal diseases that has a strong impact on people's living standard and life-span.Research data shows that sophisticated myocardial cell lacks regenerative power, and after myocardial infarction (MI) took place, the infarcted region cardiac muscle can only be by proliferation of fibrous tissue, and the scar tissue replacement by no contractile function causes heart function to descend.Lack clinically at infarcted myocardium regeneration, the essence methods of treatment of rebuilding, and the cell replacement treatment is arisen at the historic moment and is become an ideal therapeutic strategy.Mesenchymal stem cells MSCs (Mesenchymal stem cells, MSCs), it is a kind of similar fibroblastic class cell in marrow, have multi-direction differentiation potential, can transform to osseous tissue, cartilaginous tissue, skin, hemopoietic stem cell supporting dielectric, Skeletal Muscle Cell and myocardial cell.Autologous bone marrow mesenchymal stem cells also has the convenience of drawing materials, non-immunogenicity, has multidirectional differentiation potential, conforms with the ethics requirement and does not have tumorigenicity, has the incomparable advantage of other stem cell, therefore, inquire into external a large amount of amplifications of hMSCs and induce differentiation condition to have far reaching significance to the repairing and treating of injury of myocardium to the myocardial cell.In myocardial cell's differentiation mechanism, present induction method comprises that mainly chemical induction and tissue microenvironment induce at research MSCs.Wherein (5-azacytidine is the most frequently used 5-AZA) to chemical inducer with U-18496.5-AZA is the analogue of cytidine, thereby it can cause DNA methyl carrying out property in cell division cycle to be lost with DNA covalent attachment formation mixture, and the effect of performance demethylation starts some relevant allelic expression such as MyoD.Abroad have the scholar with the adult mice marrow stromal cell after U-18496 is induced, can produce the cell mass of autonomous myotube structure of shrinking, microscopically similar embryonic myocardium cell.Domestic also have 5 U-18496s of employing to induce the Medulla Sus domestica interstital stem cell, and cell becomes myocardial cell's form, expresses myocardium specificity contractile protein.But induce the in vitro study of differentiation not appear in the newspapers as yet for human marrow mesenchymal stem cell (hMSCs) to the myocardial cell at present, Given this, we with the hMSCs in-vitro separation, the amplification after, add 5 U-18496 chemical inductions, explore bone marrow interstital stem cell is converted into the myocardial cell under external electrochemical conditions process and method, for providing experimental basis from body adoptive therapy myocardial infarction behind the MSCs amplification in vitro.
Summary of the invention
The present invention relates to a kind of biological characteristics that utilizes mesenchymal stem cells MSCs, separate amplification adult bone bone marrow-drived mesenchymal stem, add the finite concentration chemical inducer, directional induction in vitro is divided into the method for cardiac-like muscle cell.
The purpose of this invention is to provide a kind ofly with after adult bone bone marrow-drived mesenchymal stem in-vitro separation amplification, a large amount of amplification, directional induction is divided into the method for the cardiac-like muscle cell with myocardium specificity contractile protein.For achieving the above object, the present invention is by the following technical solutions: 1) adopt density gradient centrifugation separation and Culture human marrow mesenchymal stem cell, utilize self and the multiplication characteristic of stem cell that stem cell is increased, thereby reach the purpose that obtains a large amount of stem cells.2) isolated cell is in external scheme of carrying out directional induction differentiation, it is characterized in that specific cultivation algebraically (2-6,11 cells) induces, and with specific chemical inducer U-18496, specific concentrations (5-10 μ M) is carried out directional induction; 3) isolated cell is in external scheme of carrying out the directional induction differentiation, and its feature also is the short cell proliferation of bFGF and promotes myocyte's phenotypic expression function to combine with the demethylation effect of 5-AZA, more helps people MSCs to the cardioblast directed differentiation.
The invention provides the additional source after myocardial cell injury lacks, for stem cell transplantation substitute the clinical treatment myocardial infarction and late period cardiac insufficiency etc. lay a good foundation, and for myocardial infarction and late period cardiomyopathy such as cardiac insufficiency drug screening a new model is provided.
Content of the present invention is unexposed to be delivered, and those skilled in the art can not obtain separation induction method of the present invention according to existing technology deduction as not spending creative work at all.
Embodiment
The separation of embodiment 1, mesenchymal stem cells MSCs, purifying and former generation, the cultivation of going down to posterity
Aseptic condition extracts healthy volunteer's ilium marrow down, add thorough mixing in the DMEM-F12 nutrient solution, centrifugal supernatant and the lipid layer abandoned, after adding the abundant mixing of perfect medium, bone marrow fluid is added on the Percoll parting liquid of density 1.073g/ml gently, centrifugal 30 minutes of 900g (under the room temperature) gets the above part of white corpuscle rete, adds the DMEM-F12 nutrient solution that contains 10% foetal calf serum and (adds green grass or young crops-Streptomycin sulphate 100u/ μ g.ml in advance
-1) in abundant mixing, centrifuge washing is standby, with isolated cells by 2 * 10
5/ cm
2Concentration be inoculated in the 50ml plastic culture bottle, place 37 ℃, 5%CO
2With cultivate in the incubator of saturated humidity, change nutrient solution behind the 48h, discard not attached cell, changed liquid once in later per 2～3 days.Tryptic digestion with 0.25% when cell length to 80% merges goes down to posterity, and carefully pasteur pipet is blown and beaten into behind the single cell suspension with 1 * 10
5/ ml kind is planted in new nutrient solution, changes liquid once in per 2～3 days.
Embodiment 2, the differentiation of adult bone bone marrow-drived mesenchymal stem directional induction in vitro
Get separation and Culture 3 generation descendant MSCs, use 0.25% tryptic digestion, after PBS cleans, with 2 * 10
5/ cm
2Density be inoculated in the 24 hole plastic culture plates, 5 U-18496s that in perfect medium, add 3 μ mol/L, 5 μ mol/L, 10 μ mol/L, induce differentiation after 24 hours, nutrient solution is abandoned in suction, PBS cleans twice, continue to use complete culture medium culturing, changed liquid once in 2～3 days, the tryptic digestion with 0.25% when cell length to 80% merges goes down to posterity.Induce 14,21 days the cell climbing sheet in back, fixing, immunofluorescence dyeing is observed desmin, myocardium early transcription factor GATA4, the expression of myocardium specificity cTnI and intercalated disc albumen connexin43.5.5 * 10
5Individual MSCs can obtain (2.3 ± 0.36) * 10 after 12 generations of increasing
9Individual cell, amplification is (4.6 ± 0.45) * 10 approximately
4Doubly; 5, cellular form was elongated after 5 U-18496s of 10 μ mol/L carried out chemical induction, volume increases, 20～30% cytogamy form multinuclear myotube spline structure when inducing back 14 days, the myotube structure does not appear in 3 μ mol/L group MSCs, induce in back 21 days 5,10 μ mol/L group people MSCs desmin and myocardium early transcription factor GATA4 express strong positive, myocardium specificity cTnI, and the expression of intercalated disc albumen connexin43 weak positive, cTnI positive staining cell rate 10 μ mol/L induce group to be higher than 5 μ mol/L and induce group (P＜0.05); 3 μ mol/L induce the expression feminine gender of group and negative control group cardiac muscle specific proteins.
Get former being commissioned to train respectively and support 72h cell, amplification in vitro the 2nd, 6,11 generation MSCs, with 8.0 * 10
3/ cm
3Concentration is inoculated in six orifice plates that are placed with the disinfection cap slide, the preparation cell climbing sheet, every hole adds 2ml complete culture solution (DMEM-F12+10%FBS), when reaching the 60-70% fusion, behind bFGF (100ng/ml) the pre-treatment 24h, change 5-AZA (final concentration is 10 μ moL/L) and induce 24h, the PBS washing, be replaced by the perfect medium that contains bFGF (2ng/ml), changed nutrient solution in per afterwards three days, and examine under a microscope the cellular form variation every day.Induce back 20 days cell climbing sheet, carry out the immunohistochemical methods of Troponin I (troponinI) and desmin (desmin) and identify.2nd, 6,11 generation people MSCs induce the orientable cardiac-like muscle cell that is divided into through 5-AZA and bFGF, express troponinI and desmin; RT-PCR shows: induce back cell cardiac muscle specific factor gene β-myosinogen heavy chain (β-MHC) positive expression, wherein the expression of β-MHC of the 6th generation MSCs than the 2nd, 11 generation height.
Claims (3)
1. one kind is separated amplification cultivation grownup mesenchymal stem cells MSCs (Mesenchymal stem cells, MSCs) method, it is characterized in that utilizing the stem cell physiological property, adopt the DMEM-F12+10% foetal calf serum as minimum medium, by in time, go down to posterity people MSCs is carried out purifying and amplification cultivation repeatedly, thereby obtain the purpose of a large amount of MSCs of high purity.
2. separate cell that amplification obtains in external scheme of carrying out the directional induction differentiation by claim 1, it is characterized in that with specific medicine, specific concentrations and specific cultivation algebraically (2-6 is for cell and 11 generations) carry out directional induction having under the serum condition.
3. carry out the scheme of directional induction differentiation in vivo by the isolated cell of claim 1 separation method, be characterised in that and adopt specific concentrations, the particular growth factor to handle and to combine with the demethylation effect of U-18496 (5-AZA) when inducing, help people MSCs to myocardial cell's directed differentiation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA03109287XA CN1536076A (en) | 2003-04-09 | 2003-04-09 | Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA03109287XA CN1536076A (en) | 2003-04-09 | 2003-04-09 | Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1536076A true CN1536076A (en) | 2004-10-13 |
Family
ID=34319276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA03109287XA Pending CN1536076A (en) | 2003-04-09 | 2003-04-09 | Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1536076A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373177A (en) * | 2010-08-23 | 2012-03-14 | 杨跃进 | Methodological system construction of in vitro differentiation of pig marrow mesenchymal stem cells into myocardial cells |
| CN101426902B (en) * | 2006-04-28 | 2013-03-27 | 第一三共株式会社 | Method for differentiation induction of myocardial cell from pluripotent stem cell |
| CN103087985A (en) * | 2011-11-07 | 2013-05-08 | 北京清美联创干细胞科技有限公司 | Method for inducing mesenchymal stem cells to directionally differentiate into cardiac cells |
| CN101864392B (en) * | 2005-12-13 | 2016-03-23 | 国立大学法人京都大学 | Nuclear reprogramming factor |
-
2003
- 2003-04-09 CN CNA03109287XA patent/CN1536076A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864392B (en) * | 2005-12-13 | 2016-03-23 | 国立大学法人京都大学 | Nuclear reprogramming factor |
| CN101426902B (en) * | 2006-04-28 | 2013-03-27 | 第一三共株式会社 | Method for differentiation induction of myocardial cell from pluripotent stem cell |
| CN102373177A (en) * | 2010-08-23 | 2012-03-14 | 杨跃进 | Methodological system construction of in vitro differentiation of pig marrow mesenchymal stem cells into myocardial cells |
| CN103087985A (en) * | 2011-11-07 | 2013-05-08 | 北京清美联创干细胞科技有限公司 | Method for inducing mesenchymal stem cells to directionally differentiate into cardiac cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mayo et al. | Neural crest-derived dental stem cells—where we are and where we are going | |
| Nakashima et al. | Mobilized dental pulp stem cells for pulp regeneration: initiation of clinical trial | |
| CN103667182B (en) | A kind of mesenchymal stem cells MSCs is in vitro to induction method and the inducing culture of osteoblast differentiation | |
| CN102449141B (en) | Isolation of human umbilical cord blood-derived mesenchymal stem cells | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN110564682B (en) | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes | |
| CN112292447B (en) | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| CN101285053A (en) | Process for coculturing cord blood hematopoietic stem cells and mesenchymal stem cells in dynamic suspending condition | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| KR20190112186A (en) | Method and system for regenerating hair using 3D organoid system of hair follicle stem cells | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| Huang et al. | Proliferation and odontogenic differentiation of human umbilical cord mesenchymal stem cells and human dental pulp cells co-cultured in hydrogel | |
| CN101045916A (en) | Follicular stem cell originated from human hair and its amplifying prepn process | |
| Ingavle et al. | Mimicking megakaryopoiesis in vitro using biomaterials: Recent advances and future opportunities | |
| Sándor et al. | Combining adipose-derived stem cells, resorbable scaffolds and growth factors: An overview of tissue engineering | |
| CN103497892A (en) | Cell culture substrate, and preparation method and application thereof | |
| CN108324993B (en) | Stem cell complex for inducing hair regeneration, preparation method and application thereof | |
| CN1778905A (en) | Separating culture and use for fatty mesenchymal dry cell | |
| Lu et al. | Hematopoietic stem cells: ex-vivo expansion and therapeutic potential for myocardial ischemia | |
| CN103387959A (en) | Stem cell from deciduous teeth, and preparation method and application thereof | |
| CN1536076A (en) | Method for making adult bone marrow mesenchymal stem cells implement in vito amplification and directionally inducing them to be differentiated into myocardium-like cells | |
| CN101525596A (en) | Method for obtaining stem cell by using serum of same cord blood | |
| Li et al. | Adipose-derived mesenchymal stem cell seeded Atelocollagen scaffolds for cardiac tissue engineering | |
| CN109402049A (en) | A kind of preparation method for the tissue engineering fat stem cell lamella with cartilage differentiation potential can be used for cartilage damage reparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |