CN101709289A - Method for inducing transformation of totipotent stem cells into mesenchymal stem cells - Google Patents
Method for inducing transformation of totipotent stem cells into mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a method for inducing the transformation of totipotent stem cells into mesenchymal stem cells. In the method, a simple culture method, a fibroblast medium and a monoclonal separation method are adopted to purify and obtain mesenchymal stem cells, the mesenchymal stem cell content is over 95 percent, and more than 109 mesenchymal stem cells can be obtained. The invention has the advantages that: (1) the cells obtained by the method have excellent boundary expansion performance, have properties that can be retained in 15 generation and can from bones, cartilage and muscle tendons; (2) the method for acquiring the cells of the invention is simple, high in efficiency and free from flow type, magnetic bead and the like sorting and purification methods and greatly reduces the cost of the transformation of totipotent stem cells into mesenchymal stem cells; and (3) the method is suitable to be used to repair of defects in tissues such as bone, muscle tendon, cartilage and skin and suitable to be used as seed cells in tissue engineering.
Description
(1) technical field
The present invention relates to a kind of method of inducing myeloid-lymphoid stem cell to become mescenchymal stem cell.
(2) background technology
The damage of the various tissues of human body is very general, and especially as connective tissue damages such as bone, cartilage defect and tendon, ligament more and more (account for sport injury 50%), and skin injury is countless especially.Have data to show, in calendar year 2001, the bone transplantation in Europe has 408000 examples, and just there are 605000 examples in single U.S..At present, population every year of global over-65s is with 2～3% speed increment.And since the raising of the change of the raising of people's living standard, mode of life and medical science level these all caused to frontalis tendon, ligament, cartilage, bone transplant, more demands of bone prosthesis and using widely.
In China, according to incompletely statistics, the whole nation is annual because of factors such as all kinds of traffic accidents, orthopaedic diseases, causes bone patient damaged or bone injury that 3,000,000 people are arranged, the unsound number of bone has up to ten million, and the market total value of the annual individual coupling bone in the whole nation is at least more than 50,000,000 yuan according to estimates.Tendon injury is one of modal motional injury, accounts for it more than 50%.After the tendon rupture, corresponding joint loses movable function.Statistics shows, has up to ten million tendon injury cases in per 200,000,000 populations in 1 year at least.Owing to the change of mode of life, the hand tendon injury case that life-time service computor-keyboard and conveying mobile phone news in brief etc. causes also increases with the speed in every year 5% in addition.
Tissue defects such as clinical locomotor damage at present and skin are perhaps repaired by nondegradable biomaterial mainly by repairing reinforcement from body/allosome tissue.But these methods of treatment all have its inherent defective.Need to sacrifice the function that supplies the district as the autologous transplanting tissue, and supply is very limited; Allosome tissue's source difficulty and may have problem on immunity and the pathology.
The present method of tissue engineering method that application prospect is arranged most of tissue defects such as locomotive organ damage and skin that is considered to regenerate, the application of Method of Tissue Engineering regeneration defective tissues is explored in existing many at present researchs.According to authoritative magazine lancet, the successful patient who is applied to trachea defect of tissue engineering trachea provides foundation for organizational project is applied to tissue regeneration recently.Organizational project be compound seed cell, material and somatomedin in vivo or external structure functional organization is used to repair or the method for the defective tissue of regenerating.Seed cell is a fundamental element important in the organizational project, is the basis that the tissue that makes up has function and internal regeneration defective tissue.
Being applied to reticular tissue regenerated cell at present mainly contains various tissue-derived adult mescenchymal stem cells, skin flbroblast etc. and is divided into ripe somatocyte and myeloid-lymphoid stem cell (embryonic stem cell and from the somatocyte source induce myeloid-lymphoid stem cell (iPS)).But all there is its inherent defective in these cell sources: 1) somatocyte of differentiation and maturation only has limited differentiation and amplification ability.2) mescenchymal stem cell has stronger differentiation and amplification ability, yet still fails to reach the ability of the holomorphosis of similar embryonic tissue.3) embryonic stem cell is an embryonic cell the earliest, has all-round differentiation capability, the cell that is considered to have most regeneration potential, however because it has very strong tumorigenicity, undifferentiated embryonic stem cell can't be applied to repair.4) the similar embryonic stem cell of iPS, undifferentiated iPS can't be applied to repair.But can be by autogenous cell, so application prospect is widely arranged.But tissue regeneration potential in view of embryonic stem cell and iPS, as embryonic stem cell or iPS directional induction are divided into reticular tissue stem cell or precursor cell (progenitor cells such as mescenchymal stem cell and bone, cartilage, tendon), remove its tumorigenicity, just can make myeloid-lymphoid stem cell be applied to the organizational project reparation.It is comparatively loaded down with trivial details and cost is higher at present myeloid-lymphoid stem cell to be induced into the mescenchymal stem cell method, needs to use method purifying cells such as streaming.Therefore, seeking a kind of easy low cost and do not add compositions and methods in addition and induce myeloid-lymphoid stem cell to become mescenchymal stem cell, is that myeloid-lymphoid stem cell is applied to the reticular tissue engineering prerequisite is provided.
(3) summary of the invention
The object of the invention provides a kind of myeloid-lymphoid stem cell of inducing simply, efficiently becomes the method for mescenchymal stem cell.
The technical solution used in the present invention is:
A kind of method of inducing myeloid-lymphoid stem cell to become mescenchymal stem cell, described method comprises:
(1) (treatment process is referring to " preparation of the cultivation of mouse embryo stem cell---feeder layer " myeloid-lymphoid stem cell to be incubated at the mouse MEF feeder layer that mitomycin handled, cytobiology magazine Chinese Journal of Cell Biology 2009,31 (2): 291-292), cultivated 3～5 days with cell culture medium I, it (is pancreatin/EDTA Digestive system with pancreas enzyme-EDTA that myeloid-lymphoid stem cell covers with the back, pancreas enzyme concentration 0.01～0.05%, EDTA concentration 0.01～0.05% w/v) is digested to single cell suspension; Described cell culture medium I final concentration is composed as follows: 5～20% (w/v, the quality concentration of volume percent, contain this component 1g in certain concentration of component 1% expression 100mL nutrient solution) the KO serum substitute, 1～5mM L-glutaminate,～5.0% 0.5 (w/v) non-essential amino acid, 5～20ng/ml Basic Fibroblast Growth Factor, 2,50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are KO-DMEM;
(2) step (1) single cell suspension is with 100～1000cell/cm
2Density is planted on culture dish, the cultivation of going down to posterity in cell culture medium II; Described cell culture medium II final concentration is composed as follows: 10～30% (w/v) foetal calf serum, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM;
(3) step (2) is cultivated the third generation cell of acquisition again with 2～10cell/cm
2Density is planted on culture dish, cultivates to grow until monoclonal cell in cell culture medium III, promptly gets described mescenchymal stem cell; Described cell culture medium III final concentration is composed as follows: 5～20% (w/v) foetal calf serum, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM.
Described cell differentiates for being induced by myeloid-lymphoid stem cell, the fibroblast-like cells that under defined medium, forms through the low density plantation, tool bone, cartilage, tendon and fatty differentiation capability, with become somatocyte to compare also to have good amplification ability, it is above and keep original characteristic to be passaged to for 15 generations.
Described embryonic stem cell can adopt commercial commodity, as H1, the H9 clone of WiCell Inc company, also can obtain from discarded embryo.As follows by the concrete grammar that obtains embryonic stem cell among the embryo: 1. as to collect the 3rd day inferior quality embryo of IVF/ICSI treatment cycle, vitro culture to 5 is during by 6 days, can tell inner cell mass cultivates on mouse embryo fibroblasts, after 9～15 days the inner cell mass that grows is dispelled or trysinization, continue on l cell, to cultivate.2. pick out the single cell clone group that does not break up form, dispel into the little group of 50～100 cells existing cultivation the in back with homogeneous.3. so repeat, until becoming system.
Mescenchymal stem cell is identified: 1) cellular form is observed.Cell attachment is observed the attached cell form every day for differentiation first day, monoclonal cell screening back fibroblast-like cells ratio＞95%.2) cell phenotype is identified, and the mesenchymal cell phenotype positive (CD44, CD90, CD105, CD106), and hematopoiesis system table shape feminine gender (CD34), mesenchymal cell accounts for more than 95%.3) propagation, differentiation and tissue differentiation ability cell have bone, cartilage and fatty three is a differentiation capability.
Preferably, cell culture medium I final concentration is composed as follows in the described step (1): 10%KO serum substitute, 2mM L-glutaminate, 1% non-essential amino acid, 10ng/ml Basic Fibroblast Growth Factor 2,100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are KO-DMEM.
Preferably, cell culture medium II final concentration is composed as follows in the described step (2): 20% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.
Preferably, cell culture medium III final concentration is composed as follows in the described step (3): 10% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.
The present invention further improves on the basis of former research, utilizes simple cultural method, uses inoblast substratum and mono-clonal sorting method, is purified into mescenchymal stem cell, makes mescenchymal stem cell content more than 95%, can obtain 10
9Above mescenchymal stem cell, thus enough reticular tissue seed cells obtained.This technology will promote myeloid-lymphoid stem cell to be applied to tendon, bone, cartilage and skin tissue engineering.
Beneficial effect of the present invention is mainly reflected in:
(1) cell that the present invention obtains has good expansion border ability, can reach for 15 generations and preserving property, and have the formation bone, the ability of cartilage and tendon;
(2) it is simple that the present invention obtains the cell preparation method, and the efficient height need not streaming, and magnetic bead etc. separate purification process, greatly reduces the cost that obtains mescenchymal stem cell from myeloid-lymphoid stem cell;
(3) cell of the present invention is suitable for the defect repair of tissue such as bone, tendon, cartilage, skin and as the seed cell of organizational project.
(4) description of drawings
Fig. 1 is the adherent differentiation primary cell of myeloid-lymphoid stem cell;
Fig. 2 is the seed cell form in myeloid-lymphoid stem cell source, induces the back cellular form to have the homogeneity of height;
Fig. 3 has the mescenchymal stem cell phenotype for the seed cell in myeloid-lymphoid stem cell source;
It is differentiation capability that Fig. 4 is similar to three of mescenchymal stem cell for the seed cell in myeloid-lymphoid stem cell source has: A: bone differentiation capability, B: cartilage differentiation capability, C: fatty differentiation capability, D: tendon differentiation capability;
Fig. 5 repairs tendon for the seed cell in myeloid-lymphoid stem cell source; A: seed cell is planted the glue in fibrin; B: it is damaged that the seed cell in myeloid-lymphoid stem cell source is repaired the kneecap tendon;
Fig. 6 for the seed cell reparation in myeloid-lymphoid stem cell source after tendon; A: the structure of similar tendon appears in defect; B: continuous sophisticated collegen filament appear in defect; The normal tendon of T-, J-repairs the junction, and N-repairs the position;
Fig. 7 repairs cartilage for the seed cell in myeloid-lymphoid stem cell source; A: cartilage defect after the seed cell reparation in myeloid-lymphoid stem cell source; B: cartilage defect histology after the seed cell reparation in myeloid-lymphoid stem cell source.
Fig. 8 is damaged for the seed cell reparation bulk bone in myeloid-lymphoid stem cell source; A: the seed cell in myeloid-lymphoid stem cell source is planted in the decalcification bone and is made up tissue engineered bone; B: it is damaged that tissue engineered bone is repaired the bulk bone.
Fig. 9 is the seed cell in the myeloid-lymphoid stem cell source feeder layer cells as human embryo stem cell: human embryo stem cell can be on the seed cell in myeloid-lymphoid stem cell source normal growth.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1 (this example reagent removes specified otherwise all available from Invitrogen company):
1) culture hole is with 0.1% (w/w) gelatin bag quilt, and (human embryo stem cell, by obtaining among the discarded embryo, method is seen the summary of the invention part myeloid-lymphoid stem cell.Discarded embryo is for donating after embryo supplier and family members agree) be seeded on the mouse MEF feeder layer that mitomycin handled, usefulness cell culture medium I is in 37 ℃, 5%CO
2Cultivated 3～5 days under (residue 95% is air) condition, after cell covers with, use 0.025% pancreatin-0.05%EDTA (Invitrogen) digestion, dispel, obtain single cell suspension.
The MEF feeder layer is handled:
1. get new culturing bottle, add 0.1%Gelatin (SigmaG2500) solution and cover pre-treatment 30min, with before sopping up Gelatin solution.
2. get the MEF cell that needs processing, Mitomycin C (Sigma M0503) is added in the MEF nutrient solution mixing with the 10mg/ml working concentration.
3. put and act on 3h in the incubator.
4. inhale and abandon waste liquid.
5. wash 5 times with PBS.
6. add 0.025% Trypsin-EDTA digestion.Examine under a microscope, when small amounts of cells floating, when the crack appears in adherent cellular layer, with at the bottom of the transfer pipet piping and druming washing bottle 5～6 times.
7. add an amount of MEF complete culture solution at once and stop digestion.
8. cell suspension is transferred in the centrifuge tube, centrifugal 1000r/min, 5min.Supernatant liquor is abandoned in suction.
9. the MEF cell of handling adds the resuspended counting of an amount of nutrient solution, with 3.0 * 10
4Individual/cm
2(MEF) density is layered on the culturing bottle that gelatin (gelatin) handled equably.
10. put standing over night in the incubator, make it adherent.
Cell culture medium I final concentration is composed as follows: 10% (w/v) KO serum substitute (Invitrogen), the 2mM L-glutaminate, 1% (w/v) non-essential amino acid (Invitrogen), 10ng/ml Basic Fibroblast Growth Factor 2 (FGF2), 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are KO-DMEM.
The MEF complete culture solution: 10% (w/v) foetal calf serum, 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are high sugared DMEM.
2) single cell suspension is with 500～600cell/cm
2Density is planted on culture dish, the cultivation of going down to posterity in cell culture medium II; Described cell culture medium II final concentration is composed as follows: 20% (w/v) foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM;
3) step 2) cultivate the third generation cell (cell becomes the fibroblast-like cells than homogeneous) of acquisition again with 6～10cell/cm
2Density is planted on culture dish, cultivates to grow until monoclonal cell in cell culture medium III, promptly gets described mescenchymal stem cell; Described cell culture medium III final concentration is composed as follows: 20% (w/v) foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.See that cell is the fibroblast-like cells of homogeneous, and have mesenchyme phenotype (Fig. 1～Fig. 3).
Embodiment 2:
With the cell cultures of embodiment 1 gained after cover with, plantation 2.5 * 10
7Cell (50ul, 5 * 10
8/ ml) in chitosan gel rubber, make up tissue engineering bone/cartilage (seeing Fig. 4 B).
Embodiment 3:
With the cell cultures of embodiment 1 gained after cover with, add the 50ug/ml vitamins C and cultivate formation cell sheet after 14 days, the cell sheet (is stretched 10%) under static tension force stimulates cultivated for 2 weeks, histology and electron microscopic observation, see cell regular arrangement on the mechanics direction, the outer tendon from tissue engineering (seeing Fig. 4 D) of constituting body.
Embodiment 4:
It is damaged to be used to repair ligamentum patellae in the cell implantation rat body with embodiment 1 gained, plantation 5 * 10
5Cell (20ul, 2.5 * 10
7/ ml) in the Fibrin gel, plant in the rat ligamentum patellae.Can find the tissue defect of growing into after 4 weeks, form functional tendon tissue (seeing Fig. 5,6).
Embodiment 5:
The cell of embodiment 1 gained is implanted rat, and to be used to repair knee cartilage damaged, and 5 * 10
5Cell (20ul, 2.5 * 10
7/ ml) in collagen gel, tissue is grown into well after 4 weeks, can form functional cartilaginous tissue, does not have tangible Inflammatory response (see figure 7).
Embodiment 6:
Cell sheet and the compound structure tissue engineered bone of decalcification bone (seeing Fig. 8 A) with embodiment 3 gained are used to repair radius damaged (seeing Fig. 8 B), can promote freshman bone tissue to grow into.
Embodiment 7:
With the cell of embodiment 1 gained after mitomycin c handles as human embryo stem cell feeder layer cells (see figure 9): but the human embryo stem cell normal growth.
Claims (4)
1. method of inducing myeloid-lymphoid stem cell to become mescenchymal stem cell, described method comprises:
(1) myeloid-lymphoid stem cell is incubated on the mouse MEF feeder layer that mitomycin handled, cultivated 3～5 days with cell culture medium I, myeloid-lymphoid stem cell covers with the back and is digested to single cell suspension with pancreas enzyme-EDTA; Described cell culture medium I final concentration is composed as follows: 5～20%KO serum substitute, 1～5mM L-glutaminate, 0.5～5.0% non-essential amino acid, 5～20ng/ml Basic Fibroblast Growth Factor 2,50～200U/m1 penicillin, 50～200U/ml Streptomycin sulphate, solvent are KO-DMEM;
(2) step (1) single cell suspension is with 100～1000cell/cm
2Density is planted on culture dish, the cultivation of going down to posterity in cell culture medium II; Described cell culture medium II final concentration is composed as follows: 10～30% foetal calf serums, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM;
(3) step (2) is cultivated the third generation cell of acquisition again with 2～10cell/cm
2Density is planted on culture dish, cultivates to grow until monoclonal cell in cell culture medium III, promptly gets described mescenchymal stem cell; Described cell culture medium III final concentration is composed as follows: 5～20% foetal calf serums, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM.
2. the method for claim 1, it is characterized in that cell culture medium I final concentration is composed as follows in the described step (1): the 10%KO serum substitute, the 2mM L-glutaminate, 1% non-essential amino acid, 10ng/ml Basic Fibroblast Growth Factor 2,100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are KO-DMEM.
3. the method for claim 1 is characterized in that cell culture medium II final concentration is composed as follows in the described step (2): 20% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.
4. the method for claim 1 is characterized in that cell culture medium III final concentration is composed as follows in the described step (3): 10% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948803A (en) * | 2010-09-13 | 2011-01-19 | 中山大学中山眼科中心 | Human epidermal derived mesenchymal stem cell-like pluripotent cells and preparation method thereof |
| CN106190960A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of preparation method of the cell preparation promoting wound Regeneration and Repair |
| CN107858328A (en) * | 2017-11-15 | 2018-03-30 | 广州赛隽生物科技有限公司 | A kind of neural crest pedigree mesenchymal cell and its method of inducing differentiation from multipotential stem cell |
| CN109136191A (en) * | 2018-08-27 | 2019-01-04 | 上海市儿童医院 | A kind of Tc1iPs cell line and its construction method |
| CN110551204A (en) * | 2019-09-12 | 2019-12-10 | 深圳刚华健医疗有限公司 | Preparation method of sub-totipotent mesenchymal stem cell secretin |
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2009
- 2009-12-15 CN CN200910155482A patent/CN101709289A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948803A (en) * | 2010-09-13 | 2011-01-19 | 中山大学中山眼科中心 | Human epidermal derived mesenchymal stem cell-like pluripotent cells and preparation method thereof |
| CN101948803B (en) * | 2010-09-13 | 2012-09-05 | 中山大学中山眼科中心 | Human epidermal derived mesenchymal stem cell-like pluripotent cells and preparation method thereof |
| CN106190960A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of preparation method of the cell preparation promoting wound Regeneration and Repair |
| CN107858328A (en) * | 2017-11-15 | 2018-03-30 | 广州赛隽生物科技有限公司 | A kind of neural crest pedigree mesenchymal cell and its method of inducing differentiation from multipotential stem cell |
| CN109136191A (en) * | 2018-08-27 | 2019-01-04 | 上海市儿童医院 | A kind of Tc1iPs cell line and its construction method |
| CN110551204A (en) * | 2019-09-12 | 2019-12-10 | 深圳刚华健医疗有限公司 | Preparation method of sub-totipotent mesenchymal stem cell secretin |
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