CN101245336A - Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis - Google Patents
Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis Download PDFInfo
- Publication number
- CN101245336A CN101245336A CNA2008100200943A CN200810020094A CN101245336A CN 101245336 A CN101245336 A CN 101245336A CN A2008100200943 A CNA2008100200943 A CN A2008100200943A CN 200810020094 A CN200810020094 A CN 200810020094A CN 101245336 A CN101245336 A CN 101245336A
- Authority
- CN
- China
- Prior art keywords
- bone marrow
- stem cell
- cell
- cerebrospinal fluid
- autologous
- 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
Abstract
The invention relates to a method of the induction and differentiation of the neural stem cells and the neural cells from bone marrow stromal cells which are cultured by autologous bone marrow, autologous serum and autologous cerebrospinal fluid. The method pertains to the methods for differentiating the bone marrow stem cells into the neural stem cells. A conventional low-sugar DMEM/F 12 culture medium is adopted, the autologous serum replaces the original fetal bovine serum, the autologous cerebrospinal fluid replaces the original additive and a stimulating factor, a small amount of autologous bone marrow is used for culturing more cells with the number that can meet the clinical needs. The method has the advantages of low cost and easy material selection, avoiding zoonosis and foreign protein exclusive reaction, and so on. The method of inducing the bone marrow stromal cells into the neural stem cells by using cerebrospinal fluid can effectively solve the shortcomings of the traditional methods.
Description
Technical field
The present invention relates to the method that a kind of bone marrow stem cell is divided into neural stem cell.Specifically be a kind of by autologous bone marrow, autoserum, cultivate the method that bone marrow stromal cell is induced to differentiate into neural stem cell, neuronal cell from body cerebrospinal fluid.
Background technology
At present, bone marrow stromal cell being cultivated into bone marrow stem cell both at home and abroad adds 10% tire ox or uses low sugar DMEM/F with low sugar DMEM cultivation usually
12Add various nutritive substance serum-free culture, the former harvested cell is many but can't avoid the rejection of infecting both domestic animals and human disease, foreign protein; Latter's harvested cell amount is few, needs donor marrow amount many.State, inside and outside bone marrow stromal cell induce the method that becomes neural stem cell to be: with neurobasal and B
27As substratum, in culturing process, add people's epidermis stimulating factor (EGF) then, Prostatropin (bFGF) effect inducible factor is induced bone marrow stromal cell becomes neural stem cell.The shortcoming of this traditional method is: culture cycle is long; The expense costliness.
Summary of the invention
Discover neural factor, platelet-derived growth factor (PDGF), rhIGF-1 (IGF), sugar, albumen, polypeptide, multiple somatomedin such as the Basic Fibroblast Growth Factor, Brain Derived Neurotrophic Factor, the glial cell line-derived neurotrophic factor that not only contain multiple brain source property in the cerebrospinal fluid; Also contain intrinsic regulation and control cell differentiation of nerve cord material, as alkaline spiral-ring spiral (basic helix-100p-helix, bHLH) transcripton is the functional gene that determines cell differentiation of nerve cord; Cancer suppressor gene PTEN under normal circumstances has inhibition NSC
sThe effect of propagation; The Notch gene is an important controlling gene of determining neuronal quantity in the central nervous system growth course, and it can also keep NSC
sUndifferentiated state.That is to say in the cerebrospinal fluid to also have regulatory factor, can suppress the generation that the cell differentiation of nerve cord transition causes glioma cell except having somatomedin.Theoretically, cerebrospinal fluid is fit to the brain cell growth, safeguards enough growth conditionss, therefore utilizes cerebrospinal fluid to replace the cytokine induction method.Can solve the safety issue that present animal derived cytokine induction method exists with this novel method.
The object of the present invention is to provide a kind of bone marrow stem cell directed differentiation that can make is the novel method of neural stem cell, for human neural stem cells transplantation technology is opened up new approach.And have with low cost, draw materials easily, avoid the infecting both domestic animals and human disease, advantages such as foreign preteins rejection.The method that adopts cerebrospinal fluid to induce bone marrow stromal cell to become neural stem cell efficiently solves the defective of traditional method.
The present invention realizes with following technical scheme: adopt autoserum to substitute original foetal calf serum, substitute original additive and stimulating factor from body cerebrospinal fluid, the autologous bone marrow that takes a morsel is turned out cell quantity more, that can meet clinical needs.
Concrete preparation process is as follows:
1, the conventional low sugar DMEM/F that uses
12Substratum+10% autoserum, add the two anti-of convention amount;
2, get marrow 15ml,, give a baby a bath on the third day after its birth time with phosphate buffered saline buffer PBS with lymphocyte separation medium isolated cell layer;
3, get 1 * 10
7Cell suspension inoculation is in the cell bottle that 25ml is crossed by the poly-lysine bag;
4, change half amount in three days, change the full dose substratum after five days;
5, CD34 CD45 (bone marrow stem cell antibody) antibody cells were tested by flow cytometry 100% positive is gone down to posterity in the back amplification of two weeks totally 5 times;
6, get bone marrow stem cell 2 * 10
7Cell suspension 2ml wears cell suspension is injected subarachnoid space by doing waist, takes out 2～3ml cerebrospinal fluid before injection, and the centrifuging and taking supernatant liquor is that the cerebrospinal fluid packing is standby;
7, will pass the bone marrow stem cell in 5 generations and cover three days, CD90 CD45 (neural stem cell antibody) antibody cells were tested by flow cytometry 100% positive with cerebrospinal fluid;
8, add 2-3ml DMEM/F
12Serum free medium adds 10 μ l/L cerebrospinal fluid every day, is divided into neuronal cell about 3 days.
The present invention compared with prior art has the following advantages:
1, autologous bone marrow, autoserum can be avoided the infecting both domestic animals and human disease, the foreign preteins rejection.
2, cerebrospinal fluid itself provides enough trophic nerve stem cell, neuronal cell material and regulation and control substance, neural stem cell can be converted into neuronal cell and can prevent that again excessively differentiation from causing the generation of glioma.
3, with low cost, effective, method is simple, easy to operate, repeatable strong.
4, be a kind of novel cultural method, opened up the long-range prospect and the economic results in society of transplantation for suffering from the sacred disease patient.
5, avoid morality and ethics, alleviate patient's economical load.
Embodiment
Further specify method of the present invention and effect below by embodiment
Embodiment
1, the conventional low sugar DMEM/F that uses
12Substratum+10% autoserum, add-on adds penicillin-Streptomycin sulphate routinely;
2, get marrow 15ml,, give a baby a bath on the third day after its birth time with phosphate buffered saline buffer PBS with lymphocyte separation medium isolated cell layer;
3, get 1 * 10
7Cell suspension inoculation is in the cell bottle that 25ml is crossed by the poly-lysine bag;
4, change half amount in three days, change the full dose substratum after five days;
5, the back amplification of two weeks is gone down to posterity and is changed into bone marrow stem cell totally 5 times.(groupization of exempting from service and CD34, CD44 positive rate 100%);
6, get 2 * 10
7Cell suspension 2ml wears cell suspension is injected subarachnoid space by doing waist, takes out 2～3ml hydrocrania before injection, and the packing of centrifuging and taking serum is standby.
7, will pass the bone marrow stem cell in 5 generations and change into neural stem cell in three days with the hydrocrania covering.(immunohistochemical methods and CD90, CD45 positive rate 100%)
8, add 3ml DMEM/F
12Serum free medium adds the 10ul/L hydrocrania every day, is divided into neuronal cell about 3 days.
9, by waist wear with neural stem cell, neuronal cell once more infusion observe to have or not in subarachnoid space and repel reflection, be 10 routine patients altogether and all do not had rejection.
Case: the Meng XX, man 65 years old.Patient's cerebellar hemorrhage, stupor, urinary incontinence, the patient is progressively clear-headed after the routine treatment, but right side quadriplegia, muscular strength II, hypermyotonia, ankle clonus (+), patellar clonus (+) is in back 4 months capable neural stem cells transplantations of morbidity, all patient's muscular tension obviously descend behind the first set grafting, and muscular strength increases; It is normal to transplant back one all patient's muscular tension for the second time, and ankle clonus and patellar clonus disappear, the right IV level of Myodynamia recovery.
Claims (1)
1. a cerebrospinal fluid inducing self-body bone marrow stem cell is divided into the method for neural stem cell, it is characterized in that by autologous bone marrow, autoserum, cultivates bone marrow stromal cell from body cerebrospinal fluid and be induced to differentiate into neural stem cell, neuronal cell, and concrete steps are as follows:
1) the conventional low sugar DMEM/F that uses
12Substratum+10% autoserum adds the two anti-of convention amount;
2) get marrow 15ml, separate with lymphocyte and answer the isolated cell layer, give a baby a bath on the third day after its birth time with phosphate buffered saline buffer PRS;
3) get 1 * 10
7Cell suspension inoculation is in the cell bottle that 25ml is crossed by the poly-lysine bag;
4) change half amount in three days, change the full dose substratum after five days;
5) bone marrow stem cell antibody CD34, CD45 cells were tested by flow cytometry 100% positive is gone down to posterity in the back amplification of two weeks totally 5 times;
6) get bone marrow stem cell 2 * 10
7Cell suspension 2ml wears cell suspension is injected subarachnoid space by doing waist, takes out 2～3ml cerebrospinal fluid before injection, and the centrifuging and taking supernatant liquor is a cerebrospinal fluid, and packing is standby;
7) will pass the bone marrow stem cell in the 5th generation and cover three days, CD90, CD45 neural stem cell antibody cells were tested by flow cytometry 100% positive with cerebrospinal fluid;
8) add 2-3ml DMEM/F
12Serum free medium adds 10 μ l/L hydrocranias every day, is divided into neuronal cell about 3 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100200943A CN101245336A (en) | 2008-03-20 | 2008-03-20 | Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100200943A CN101245336A (en) | 2008-03-20 | 2008-03-20 | Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101245336A true CN101245336A (en) | 2008-08-20 |
Family
ID=39946016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100200943A Pending CN101245336A (en) | 2008-03-20 | 2008-03-20 | Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101245336A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015056258A3 (en) * | 2013-10-14 | 2015-07-09 | Hadasit Medical Research Services & Development Limited | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| CN107132089A (en) * | 2017-04-28 | 2017-09-05 | 遵义医学院附属医院 | A kind of preparation method of medical cerebrospinal fluid quality-control product |
| JP2021525106A (en) * | 2018-06-01 | 2021-09-24 | ヘルプ・ステム・セル・イノベイションズ・カンパニー・リミテッドＨｅｌｐ Ｓｔｅｍ Ｃｅｌｌ Ｉｎｎｏｖａｔｉｏｎｓ Ｃｏ．， Ｌｔｄ． | Method for producing differentiation medium and oligodendrocyte progenitor cells |
-
2008
- 2008-03-20 CN CNA2008100200943A patent/CN101245336A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015056258A3 (en) * | 2013-10-14 | 2015-07-09 | Hadasit Medical Research Services & Development Limited | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| CN105814196A (en) * | 2013-10-14 | 2016-07-27 | 哈达斯特医疗研究服务和开发有限公司 | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| US10336985B2 (en) | 2013-10-14 | 2019-07-02 | Hadasit Medical Research Services & Development Limited | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| CN107132089A (en) * | 2017-04-28 | 2017-09-05 | 遵义医学院附属医院 | A kind of preparation method of medical cerebrospinal fluid quality-control product |
| JP2021525106A (en) * | 2018-06-01 | 2021-09-24 | ヘルプ・ステム・セル・イノベイションズ・カンパニー・リミテッドＨｅｌｐ Ｓｔｅｍ Ｃｅｌｌ Ｉｎｎｏｖａｔｉｏｎｓ Ｃｏ．， Ｌｔｄ． | Method for producing differentiation medium and oligodendrocyte progenitor cells |
| JP7114134B2 (en) | 2018-06-01 | 2022-08-08 | ヘルプ・ステム・セル・イノベイションズ・カンパニー・リミテッド | Differentiation medium and method for producing oligodendrocyte progenitor cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2528250C2 (en) | Method for producing mesenchymal stem cells of human pluripotent stem cells, and mesenchymal stem cells produced by this method | |
| CN104805054B (en) | Stem cell serum-free culture medium | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN102978156A (en) | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium | |
| CN102839154A (en) | Neural stem cell culture amplification method and used culture medium | |
| KR102573180B1 (en) | Methods for preparing differentiation media and oligodendrocyte precursors | |
| CN103087977A (en) | Culture solution for in vitro efficient amplification of animal cells and application of culture solution | |
| CN102191217A (en) | Method for inducing differentiation from human umbilical cord mesenchymai stem cells (hucMSCs) into neural cells | |
| CN103881971B (en) | Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells | |
| CN107475188A (en) | A kind of cultural method of cell culture medium and embryonic stem cell | |
| Yang et al. | A simple and efficient method for deriving neurospheres from bone marrow stromal cells | |
| CN104212762B (en) | Method for culture of urine-derived pluripotent stem cells by virtue of in vitro small molecule induction | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN105132360A (en) | Method for inducing placenta-derived mesenchymal stem cells to be differentiated into islet-like cells | |
| CN1778905B (en) | Separating culture and use for fatty mesenchymal dry cell | |
| CN101831403B (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN101245336A (en) | Method for inducing self-body stem cell differentiation to be nerve stem cell with liquor cerebrospinalis | |
| CN107513519B (en) | Culture method of Schwann cells | |
| CN105886462A (en) | Composition ADSCs for ADSCs culture and ADSCs culture method | |
| CN107384864A (en) | Cell culture fluid and its application method by umbilical cord mesenchymal stem cells induction into NSC | |
| CN105087475B (en) | A kind of method that cell culture fluid and its application and induction dental pulp stem cell break up to neural-like cells | |
| CN102703380B (en) | Sub-totipotent stem cell, preparation method and application thereof | |
| CN103013918A (en) | Method for separating and subculturing primary neural stem cell of human embryo midbrain | |
| CN1920010A (en) | Method of separating multipotent adult progenitor cells from umbilical cord blood |
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 |
Open date: 20080820 |