CN101186901A - Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell - Google Patents
Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell Download PDFInfo
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- CN101186901A CN101186901A CNA2007100308826A CN200710030882A CN101186901A CN 101186901 A CN101186901 A CN 101186901A CN A2007100308826 A CNA2007100308826 A CN A2007100308826A CN 200710030882 A CN200710030882 A CN 200710030882A CN 101186901 A CN101186901 A CN 101186901A
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Abstract
The invention provides a process for inducing mescenchymal stem cell of human fetal bone marrow to differentiate to be islet beta cells. The process provided by the invention comprises the following steps including separating, purifying and augmenting mescenchymal stem cell of human fetal bone marrow, and then inducing mescenchymal stem cell of human fetal bone marrow to differentiate to islet beta cells, and beta cell mass is achieved after six to twelve days of inducement. The process provided by the invention induces mescenchymal stem cell of human fetal bone marrow to be islet beta cells which can secrete insulin in vitro. The achieved islet beta cells can be further used for preparing medicament which is used for treating diabetes. The study of the field has wide clinical application prospect and can bring relatively great economic and social benefit.
Description
Technical field
The present invention relates to cytodifferentiation, be specifically related to a kind of method of inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation.
Background technology
China is second diabetes group of people at high risk big country after India.Treated diabetes, taking medicine and insulin injection is the method for using always in the past, but takes medicine and insulin injection not only can bring heavy economical load, and can cause severe complications; In recent years, islet transplantation treatment diabetes first meeting curative effect, but be faced with tissue-derived deficiency and immunological rejection two hang-ups.Stem-cell research has brought new hope to the diabetic subject, wherein the multidirectional differentiation potential of embryonic stem cell is the strongest, almost can be divided into all adult tissue's cells that comprise pancreatic beta cell, but it is faced with ethical dispute, and have tumorigenicity and stronger immunogenicity, these effects limit its clinical application; Pancreatic stem cells is that the adult stem cell of potential to the β cytodifferentiation arranged most, but it is difficult to separate, increase and have stronger immunogenicity.Adult bone mesenchymal stem cells (mesenchymal stem cells, MSCs) carry out in-vitro separation, amplification and purifying easily, and can take from the patient from body, can solve cell source and immunological rejection problem, but its efficient to the pancreatic beta cell differentiation is lower, and the amount of insulin secretion of noble cells is few and unstable.
Summary of the invention
For overcoming the above-mentioned shortcoming and defect of prior art, the invention provides a kind of method of inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation.The method of inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation provided by the present invention may further comprise the steps:
1, the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation is characterized in that, may further comprise the steps:
(1) separation, purifying and amplification human embryo mesenchymal stem cells: separation of human embryo mesenchymal stem cells, collecting cell is cultivated then, when cell reaches 70%～80% fusion, after the D-PBS cleaning, handle with pancreatin, add perfect medium then and stop digestion, blow and beat into single cell suspension, abandon supernatant after centrifugal, again with perfect medium suspension cell again; The cultivation of going down to posterity then;
(2) inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation: suppress cell proliferation with pre-induced liquid, when treating that it reaches 90% fusion, conventional digestion is inoculated then, is induced, and induces promptly to get the islet beta-like cell mass in 6～12 days.
Step (1) is described handles preferred following process with pancreatin: adding mass concentration is 0.25% pancreatin, outwell behind the submergence all cells, adding mass concentration again is 0.25% pancreatin, goes major part, the only surplus pancreatin that just covers cell is at 37 ℃ of digestion 1～5min.
The described perfect medium of step (1) preferably contains the DMEM/F12 substratum of 100mL/L FCS, 2mmol/L glutamine 20mmol/L HEPES.
Step (1) is described to go down to posterity to cultivate and is preferably as follows process: go down to posterity first and go down to posterity in 1: 2 ratio, later in the cultivation of going down to posterity of 1: 3 ratio.
The described pre-induced liquid of step (2) preferably contains 50mL/L FCS, 2 * 10
-5Mol/L LY294002,1 * 10
-2The DMEM/F12 substratum of mol/L β-Me.
The described inoculation of step (2), induce the process of being preferably as follows: be inoculated in 25cm in 1: 2 ratio
2In the culturing bottle, every bottle adds the 5mL induced liquid and induces.Described induced liquid preferably contains the IMDM nutrient solution of 10ng/ml bFGF, 10ng/ml EGF, 1%-2%FCS.
People's tire of the present invention is meant dead stripped fetus, the MSCs of fetal period is because multiplication capacity is stronger, plasticity-is better, and immunogenicity is low, there is not into knurl, thereby have significant superiority, at the external islet beta-like cell that is the energy excreting insulin with its first one-step inducing, the islet beta-like cell can be further used for preparing the medicine for the treatment of diabetes.Therefore, the research in this field has broad clinical application prospect, can bring bigger economic benefit and social benefit.
Description of drawings
Fig. 1 is inductive MSCs aspect graph not.
Fig. 2 is the insulin-like cell group aspect graph of inducing gained after 6 days.
Fig. 3 is the negative expression figure of the Regular Insulin of not inductive MSCs; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is insulin-like cell karyon figure; Figure C is the close figure of figure A with figure B.
Fig. 4 is the Regular Insulin positive expression figure (cell climbing sheet) that induces the islet beta-like cell mass of back gained; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is islet beta-like cell karyon figure; Figure C is the close figure of figure A with figure B.
Fig. 5 is the Regular Insulin positive expression figure (paraffin section of inducing the islet beta-like cell mass of back gained; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is islet beta-like cell karyon figure; Figure C is the close figure of figure A with figure B.
Fig. 6 is the Regular Insulin positive expression figure (paraffin section) that induces the islet beta-like cell mass of back gained.
Fig. 7 is a Ultrastructural variation comparison diagram (scanning electron microscope) before and after human fetal liver mesenchyma stem cell is induced; Wherein, figure A is the ultrastructure figure before inducing; Figure B is the ultrastructure figure after inducing.
Fig. 8 is the human fetal liver mesenchyma stem cell ultrastructure figure (transmission electron microscope) after inducing; Wherein, figure A is the ultrastructure figure after inducing; Figure B is the amplification of figure A.
Fig. 9 is the electrophorogram that RT-PCR identifies the insulin-like cell group pancreas islet related gene expression after inducing.
Figure 10 is the graphic representation of different solutions to normal mouse blood sugar value influence.
Figure 11 is the graphic representation of different solutions to the influence of blood glucose in diabetic mice value.
Embodiment
The method of embodiment 1 inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation
(1) separation of human embryo mesenchymal stem cells, cultivation and amplification
Stroke-physiological saline solution flushing fetus, cutting off umbilical cord is placed in the large beaker, with volume fraction 75% alcohol-pickled 5min, aseptic taking-up fetus shin bone in the super clean bench, femur, pick the muscle that adheres to only on it, wipe out the two ends epiphysis, the 2ml syringe pump contains the Hank liquid flushing medullary space of 2%FCS, turn white up to pulp cavity, collect the liquid that washes and go in the aseptic centrifuge tube of 15ml, suction pipe is blown and beaten repeatedly with discrete cell, the centrifugal 5min of 100 * g, precipitation mostly is red corpuscle, get supernatant liquor and change another centrifuge tube (removal red corpuscle) over to, the centrifugal 5min of 500 * g abandons supernatant liquor, it is resuspended with above-mentioned Hank liquid to get sedimentary cell, make its by the 1ml syringe needle removing residual bone chip, the centrifugal 5min of 1500r/min, collecting cell is resuspended in perfect medium (DMEM/F12,10%FCS, the 2mmol/L glutamine, 20mmol/LHEPES) in, repeatedly piping and druming make cell be dispersed into single cell suspension, get 15 μ L and be used for cell counting and cell viability mensuration, by 3 * 10
5/ cm
2Be inoculated in 25cm
2In the culturing bottle, at 37 ℃, 5%CO
2Incubator in cultivate.Change nutrient solution first after inoculating 2 d, discard not attached cell.Per 2～3 d change liquid 1 time later on.When cell reached 70～80% fusions, after the D-PBS cleaning, adding the 1ml mass concentration was 0.25% pancreatin, outwell behind the submergence all cells, adding the 1ml mass concentration again is 0.25% pancreatin, goes major part, and only surplus a small amount of pancreatin covers cell, at 37 ℃ of digestion 1～5min, observation effect degree under the mirror in time adds fresh full nutrient solution and stops digestion, and suction pipe is is softly blown and beaten into single cell suspension, abandon supernatant behind the centrifugal 10min of 400g, with full nutrient solution suspension cell again.Go down to posterity first and go down to posterity, later in the cultivation of going down to posterity of 1: 3 ratio in 1: 2 ratio.
(2) inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation
Because fetus tire bone marrow MSCs multiplication capacity is very powerful, carry out the inductive cell to preparing, with pre-induced liquid (DMEM/F12,5%FCS, 20 μ mmol LY294002,10mmol β-Me) suppress its propagation, when treating that it reaches 90% fusion, conventional digestion is inoculated in 25cm in 1: 2 ratio
2In the low adhesion culturing bottle, every bottle adds 5mL induced liquid (containing 10ng/ml bFGF, the IMDM nutrient solution of 10ng/ml EGF and 1%-2%FCS), successive induction 6-12 days.Induce the insulin-like cell group of differentiation to suspend mostly or half suspension, can change nutrient solution by centrifuging.
The evaluation of embodiment 2, insulin-like cell group
(1) gross morphology changes: Fig. 1 is inductive MSCs aspect graph not.Fig. 2 is the insulin-like cell group aspect graph of inducing gained after 6 days.Be spindle shape without inductive MSCs as can be seen from Figure 1, be arranged in parallel; Induce the MSCs after 6 days to be gathered into insulin-like cell group as can be seen from Figure 2.
(2) immunocytochemical method is seen the negative expression figure of Regular Insulin that the expression of looking into Regular Insulin: Fig. 3 is not inductive MSCs; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is islet beta-like cell karyon figure; Figure C is the close figure of figure A with figure B.The immunofluorescence dyeing result shows: Regular Insulin is dyed redness by fluorescence dye Cy3, is positioned cell cytosol (figure A); Karyon is dyed by specificity nuclear dyestuff hochest33258 lining and is blue (figure B).
Fig. 4 is the Regular Insulin positive expression figure (cell climbing sheet) that induces the insulin-like cell group of back gained; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is islet beta-like cell karyon figure; Figure C is the close figure of figure A with figure B.
Fig. 5 is the Regular Insulin positive expression figure (paraffin section of inducing the insulin-like cell group of back gained; Wherein scheming A is the positive expression figure of islet beta-like cell cytosol; Wherein scheming B is that islet beta-like cell karyon reaches figure; Figure C is the close figure of figure A with figure B.
Fig. 6 is the Regular Insulin positive expression figure (paraffin section) that induces the insulin-like cell group of back gained.。
From the contrast of Fig. 3 and Fig. 4-6 as can be seen, the cell aggregation after inducing is agglomerating and be obvious Regular Insulin positive expression
(3) ultrastructure of scanning and transmission electron microscope observing insulin-like cell group
The characteristic feature of secretory cell is to have secretory granules in cell cytosol, also claims the secretion vesica; Secretory granules are being stored secretory substance, and under suitable stimulation, secretory granules can efflux the secretory substance that is comprised, and are called exocytosis; Behind exocytosis, the irregular part that is retained on the cytolemma is called microvillus.Therefore, the characteristic feature of secretory cell is to have secretion vesica and microvillus.Fig. 7 is a Ultrastructural variation comparison diagram (scanning electron microscope) before and after human fetal liver mesenchyma stem cell is induced; Wherein, figure A is the ultrastructure figure before inducing; Figure B is the ultrastructure figure after inducing.Do not induce the no vesica shape projection in MSCs surface under the scanning electron microscope as can be seen by Fig. 7 (A), and Fig. 7 (B) shows that the inductive insulin-like cell rolls into a ball surface arrangement the vesica spline structure that differs in size is arranged; Fig. 8 is the human fetal liver mesenchyma stem cell ultrastructure figure (transmission electron microscope) after inducing; Wherein, figure A is the islet beta-like cell ultrastructure figure after inducing; Figure B is the enlarged view of figure A.Can be observed cell surface under the transmission electron microscope has short and thick microvillus, has in a large number in the cell and differs in size, deep mixed vesica.Illustrate that institute's inductive cell has tentatively possessed the morphological specificity of secretory cell.
(4) identify the Regular Insulin Expression of Related Genes of inducing back insulin-like cell group with RT-PCR
β-the actin that used the Primer5.0 software design, pdx-1, insulin, glut-2, glucagon, ngn3, nestin, primers such as isl-1, company is synthetic by match Parkson, Beijing, sequence following (each sequence table can referring to SEQ No.1-16)
| Gene | Upstream primer | Downstream primer | Annealing temperature | Amplification length |
| β-actin | 5’-CCAAGGCCAACCGCGAGAAGAT GAC-3’ | 5’-AGGGTACATGGTGGTGCCGC CAGAC-3’ | 60℃ | 592bp |
| glut-2 nestin ngn3 glucagon isl-1 insulin hIns： | 5′-GCAGCTGCTCAACTAATCAC-3′ 5′-AGGATGTGGAGGTAGTGAGA-3′ 5′-AGACGACGCGAAGCTCACC-3′ 5′-CCCAAGATTTTGTGCAGTGGTT-3′ 5′-ACACATCTTTGGGGGAAAAG-3′ 5′-CATCAGAAGAGGCCATCA-3′ 5′-GCC TTT GTG AAC CAA CAC CTG-3′， | 5’-TCAGCAGCACAAGTCCCACT -3′ 5′-TGGAGATCTCAGTGGCTCT-3′ 5′-AAGCCAGACTGCCTGGGCT-3′ 5′-GCGGCCAAGTTCTTCAACAAT -3′ 5′-AAAAAGCGCAGGAAGAGAG- 3′ 5′-TGGTTCAAGGGCTTTATTCC-3 ′ 5′-GTT GCA GTA GTT CTC CAG CTG-3′ | 57℃ 55℃ 60℃ 57℃ 55℃ 53℃ 55℃ | 785bp 251bp 286bp 221bp 347bp 444bp 261bp |
RT-PCR qualification result demonstration inducing cell is expressed and is highly expressed hlns, isl-1, insulin, glut-2 and glucogan, weak expression nestin and ngn3.Fig. 9 is the electrophorogram that RT-PCR identifies the insulin-like cell group pancreas islet related gene expression after inducing.Hlns wherein, isl-1, insulin are the genes that the beta Cell of islet characteristic is expressed; GLUT-2 is a kind of in the glucose transporter family, on the beta Cell of islet film, expresses, and be beta Cell of islet blood sugar regulation important function albumen; Nestin and ngn3 have expressed in the pancreas islet precursor cell; Synthetic and the secretion glucogan of alpha Cell of islet; Therefore, uniting of these several genes expressed the characterization of molecules that explanation inductive cell has tentatively possessed beta Cell of islet and alpha Cell of islet.
(5) utilize chemiluminescence immunoassay to detect insulin secretion level in nutrient solution supernatant and the cell pyrolysis liquid
Get simple induced liquid, do not induce MSCs the nutrient solution supernatant, induce 6 d to form the nutrient solution supernatant of insulin-like cell group, detect insulin content in each supernatant with chemiluminescence immunoassay.The result does not detect less than Regular Insulin in the culture supernatant of inductive MSCs; And, can detect the Regular Insulin of (210 ± 35.07) μ U/mL through inductive insulin-like cell group; The ratio of Regular Insulin and total protein of cell is (132.7 ± 33.23) ng/mg albumen in the cell pyrolysis liquid.The inductive cell mass can be secreted after containing the serum free medium effect 2h of 5.5mmol/L glucose. the Regular Insulin of (1.3 ± 0.04) μ U/mL; And after containing the serum free medium 2h of 25mmol/L glucose, can secrete (5.7 ± 0.21) μ U/mL Regular Insulin, its stimulation index is 4.38, illustrates that cell after inducing stimulates sugar and has certain reaction and (annotate: amount of insulin secretion after stimulation index=height sugar stimulates back amount of insulin secretion/low sugar to stimulate).
Get physiological saline, simple induced liquid, induce nutrient solution supernatant (it is 3U/mL that chemoluminescence method detects insulin level) and each 300 μ L of standard insulin solutions (4U/mL) of 6 days, abdominal injection is gone in normal mouse and the diabetic mice body, and table one, different solutions are to the influence of normal mouse blood sugar value.Table two, different solutions are to the influence of blood glucose in diabetic mice value.The result shows that the standard insulin solutions all has fast and the obvious functions of blood sugar effect normal mouse and diabetic mice, and induce 6 days nutrient solution supernatant to the no blood sugar reducing function of normal mouse, but diabetic mice is had faster and the obvious functions of blood sugar effect, and effect trend is similar to the standard insulin solutions.Injection same capability physiological saline and simple induced liquid all do not have blood sugar reducing function to normal mouse and diabetic mice.
Figure 10 is the graphic representation of different solutions to normal mouse blood sugar value influence.Figure 11 is the graphic representation of different solutions to the influence of blood glucose in diabetic mice value.As seen from Figure 10, at different time points, the standard insulin solutions has rapidly and the obvious functions of blood sugar effect normal mouse, and 2.5 hours blood sugar drops to minimumly behind abdominal injection, recovers normal after 6 hours; And injecting normal saline, simple induced liquid, the nutrient solution supernatant of inducing 6 days does not significantly change normal mouse blood sugar value.As seen from Figure 11, at different time points, the standard insulin solutions has hypoglycemic activity rapidly to diabetic mice, drops to minimumly behind abdominal injection in 1.0 hours, recovers normal after 5 hours; The injection nutrient solution supernatant of inducing 6 days also has faster obvious functions of blood sugar effect to diabetic mice, drops to minimumly behind abdominal injection in 1.0 hours, recovers normal after 5 hours, and its hypoglycemic trend and time length are similar to the standard insulin solutions; And injecting normal saline, simple induced liquid, the nutrient solution supernatant of inducing 6 days does not significantly change normal mouse blood sugar value.
Table one, different solutions to the influence of normal mouse blood sugar value (X ± s, n=6, mmol/L)
| Time | The injection |
6 days nutrient solution is induced in injection | Inject simple induced liquid | Injecting normal saline |
| 0 h 1.0h 1.5h 2.5h 3h 3.5h | 6.2 2.7 1.9 1.3 2.2 3.2 | 6.63 7.4 6.46 6.83 7.1 6.9 | 6.25 8.05 7.2 6.2 7.1 6.7 | 6.7 6.5 6.8 7.3 7.5 7 |
| 4.5h 5.5h 6.5h | 5.15 7.25 6.3 | 6 7.4 6.2 | 6.9 7.3 6.6 | 6.8 7.1 7.3 |
Table two, different solutions to the influence of blood glucose in diabetic mice value (X ± s, n=6, mmol/L)
| Time | The injection |
6 days nutrient solution is induced in injection | Inject simple induced liquid | Injecting normal saline |
| 0h 0.2h 0.5 |
24.95 13.9 4.1 3.85 5.85 14.15 16 28.45 | 25.06 16.38 11.84 14.26 15.8 18.5 14.5 29.65 | 26.1 26.6 27.2 27.5 26.9 27.8 27.2 28.8 | 24.67 24.79 24.98 25.12 25.74 24.97 26.32 27.47 |
SEQUENCE LISTING
＜110〉Ji'nan University
＜120〉method of inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation
<160>16
<170>PatentIn version 3.3
<210>1
<211>25
<212>DNA
＜213〉β-actin upstream primer
<400>1
ccaaggccaa ccgcgagaag atgac 25
<210>2
<211>25
<212>DNA
＜213〉β-actin downstream primer
<400>2
agggtacatg gtggtgccgc cagac 25
<210>3
<211>20
<212>DNA
＜213〉glut-2 upstream primer
<400>3
gcagctgctc aactaatcac 20
<210>4
<211>20
<212>DNA
＜213〉glut-2 downstream primer
<400>4
<210>5
<211>20
<212>DNA
＜213〉nestin upstream primer
<400>5
<210>6
<211>19
<212>DNA
＜213〉nestin downstream primer
<400>6
tggagatctc agtggctct 19
<210>7
<211>19
<212>DNA
＜213〉ngn3 upstream primer
<400>7
agacgacgcg aagctcacc 19
<210>8
<211>19
<212>DNA
＜213〉ngn3 downstream primer
<400> 8
aagccagact gcctgggct 19
<210>9
<211>22
<212>DNA
＜213〉glucagon upstream primer
<400>9
cccaagattt tgtgcagtgg tt 22
<210>10
<211>21
<212>DNA
＜213〉glucagon downstream primer
<400>10
gcggccaagt tcttcaacaa t 21
<210>11
<211>20
<212>DNA
＜213〉isl-1 upstream primer
<400>11
<210>12
<211>19
<212>DNA
＜213〉isl-1 downstream primer
<400>12
aaaaagcgca ggaagagag 19
<210>13
<211>18
<212>DNA
＜213〉insulin upstream primer
<400>13
catcagaaga ggccatca 18
<210>14
<211>20
<212>DNA
＜213〉insulin downstream primer
<400>14
<210>15
<211>21
<212>DNA
＜213〉hIns upstream primer
<400>15
gcctttgtga accaacacct g 21
<210>16
<211>21
<212>DNA
＜213〉hIns downstream primer
<400>16
gttgcagtag ttctccagct g 21
Claims (7)
1. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation is characterized in that, may further comprise the steps:
(1) separation, purifying and amplification human embryo mesenchymal stem cells: separation of human embryo mesenchymal stem cells, collecting cell is cultivated then, when cell reaches 70%～80% fusion, after the D-PBS cleaning, handle with pancreatin, add perfect medium then and stop digestion, blow and beat into single cell suspension, abandon supernatant after centrifugal, again with perfect medium suspension cell again; The cultivation of going down to posterity then;
(2) inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation: suppress cell proliferation with pre-induced liquid, when treating that it reaches 90% fusion, conventional digestion is inoculated then, is induced, and induces promptly to get the islet beta-like cell mass in 6～12 days.
2. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 1, it is characterized in that, the described processing with pancreatin of step (1) comprises following process: adding mass concentration is 0.25% pancreatin, outwell behind the submergence all cells, adding mass concentration again is 0.25% pancreatin, go major part, the only surplus pancreatin that just covers cell is at 37 ℃ of digestion 1～5min.
3. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 1, it is characterized in that the described perfect medium of step (1) is the DMEM/F12 substratum that contains 100ml/L FCS, 2mmol/L glutamine, 20mmol/L HEPES.
4. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 1, it is characterized in that, step (1) is described to go down to posterity to cultivate and comprises following process: go down to posterity first and go down to posterity in 1: 2 ratio, later in the cultivation of going down to posterity of 1: 3 ratio.
5. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 1 is characterized in that, the described pre-induced liquid of step (2) comprises and contains 50mL/L FCS, 2 * 10
-5Mol/L LY294002,1 * 10
-2The DMEM/F12 substratum of mol/L β-Me.
6. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 1 is characterized in that, the described inoculation of step (2), induces and comprises following process: be inoculated in 25cm in 1: 2 ratio
2In the culturing bottle, every bottle adds the 5mL induced liquid and induces.
7. the method for inducing human fetal mesenchymal stem cells into pancreatic islet beta-like cytodifferentiation according to claim 6 is characterized in that, described induced liquid comprises the IMDM nutrient solution that contains 10ng/ml bFGF, 10ng/ml EGF, 1%-2%FCS.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391982A (en) * | 2011-12-08 | 2012-03-28 | 遵义医学院附属医院 | Method for in vitro induction of human amniotic mesenchymal stem cells (hAMSCs) differentiated into insulin-secreting cells (ISCs) |
| CN101875914B (en) * | 2009-04-30 | 2012-07-04 | 四川大学华西医院 | Inducing liquid for differentiation of bone marrow mesenchymal stem cells into epithelial cells and preparation and application thereof |
| CN108795845A (en) * | 2018-06-20 | 2018-11-13 | 溯源生命科技股份有限公司 | A kind of method that placenta mesenchyma stem cell is induced to differentiate to form beta Cell of islet |
| CN112522181A (en) * | 2020-12-29 | 2021-03-19 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
| CN113481146A (en) * | 2021-06-09 | 2021-10-08 | 艾可泰科生物科技(江苏)有限公司 | Method for inducing transformation of mesenchymal stem cells into islet-like cells |
-
2007
- 2007-10-17 CN CNA2007100308826A patent/CN101186901A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875914B (en) * | 2009-04-30 | 2012-07-04 | 四川大学华西医院 | Inducing liquid for differentiation of bone marrow mesenchymal stem cells into epithelial cells and preparation and application thereof |
| CN102391982A (en) * | 2011-12-08 | 2012-03-28 | 遵义医学院附属医院 | Method for in vitro induction of human amniotic mesenchymal stem cells (hAMSCs) differentiated into insulin-secreting cells (ISCs) |
| CN108795845A (en) * | 2018-06-20 | 2018-11-13 | 溯源生命科技股份有限公司 | A kind of method that placenta mesenchyma stem cell is induced to differentiate to form beta Cell of islet |
| CN112522181A (en) * | 2020-12-29 | 2021-03-19 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
| CN112522181B (en) * | 2020-12-29 | 2023-02-17 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
| CN113481146A (en) * | 2021-06-09 | 2021-10-08 | 艾可泰科生物科技(江苏)有限公司 | Method for inducing transformation of mesenchymal stem cells into islet-like cells |
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