CN104911141A - Method for inducing iPSCs to be differentiated into pancreas islet beta cells - Google Patents
Method for inducing iPSCs to be differentiated into pancreas islet beta cells Download PDFInfo
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Abstract
The invention relates to the field of biomedicine, and provides a method for inducing iPSCs to be differentiated into pancreas islet beta cells. The method includes the following steps that iPSCs are provided, and a culture medium A is used for cultivation; when the iPSCs grow to more than 90% fusion, collagenase IV is used for digestive treatment, and the iPSCs are separated into small mass cells of iPSCs; the small mass cells are inoculated on a culture medium for cultivation, a MATRIGEL MATRIX is laid on the culture medium, when the cells grow to 55-65% fusion, the cells are cultured in the culture medium B, and cells B are obtained; the cells B are cultured in a culture medium C for cultivation, and cells C are obtained; the cells C are cultured in a culture medium D, and cells D are obtained; the cells D are cultured in a culture medium E, and the pancreas islet beta cells are obtained.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to a kind of method that iPSCs of induction is divided into beta Cell of islet.
Background technology
Diabetes are a kind of common diseases with multiple severe complication, temporarily without curing therapy.Transplantation of pancreas is the important channel for the treatment of diabetes, but donor source shortage and immune rejection problems limit it and extensively carry out.Beta Cell of islet is unique cell being responsible for transhipment, synthesis and uelralante, and Regular Insulin uniquely falls hypoglycemic hormone in body, and human peripheral blood sugar level plays important regulative.
Pluripotent stem cell (induced pluripotent stem cell, iPSCs) a kind of have the of self-replication capacity and can be divided into the stem cell of multiple target function cell under certain condition, the use of pluripotent stem cell, has become the new resources that people find beta Cell of islet surrogate gradually.But, although pluripotent stem cell can solve donorcells count issue, also need before application to be first induced to differentiate into functional insulin secretory cell.In recent years, the method in domestic and international research, iPSCs being induced to differentiate into insulin secretory cell mainly utilizes genetic engineering technique to import organs of living beings tissue by transcriptional regulator transfered cell important in beta Cell of islet growth course or directly, makes iPSCs transdifferentiation be insulin secretory cell.
How effectively to select suitable micromolecular compound or substratum, thus effective implemention iPSCs Induction of committed differentiation being islet beta-like cell, is the important topic that research iPSCs is induced to differentiate into insulin secretory cell.
Summary of the invention
The object of the present invention is to provide a kind of iPSCs of induction to be divided into the method for beta Cell of islet, be intended to solve and how pass through to select suitable micromolecular compound or substratum, iPSCs Induction of committed differentiation is the problem of islet beta-like cell by effective implemention.
The present invention is achieved in that a kind of iPSCs of induction is divided into the method for beta Cell of islet, comprises the following steps,
IPSCs is provided, and uses culture medium A to cultivate;
Until described iPSCs grow to more than 90% merge time, use collagenase IV to carry out digestion process, be separated into the little clump cells of iPSCs;
Little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells merges to 55-65%, described cell is cultivated in substratum B, obtain cell B, wherein, described substratum B is the DMEM/F12 substratum of BSA, 0.45-0.55 containing 0.18-0.22% × N2,0.45-0.55 × B27,95-105ng/mL activin A and 0.08-0.12 μM of wortmannin;
Described cell B is cultivated in culture medium C, obtains cell C;
Described cell C is cultivated in substratum D, obtains cell D;
Described cell D is cultivated in substratum E, obtains beta Cell of islet.
Induction iPSCs provided by the invention is divided into the method for beta Cell of islet, and by selecting multiple specific substratum to carry out cell cultures according to specific order, can be divided into beta Cell of islet by efficiently and directionally induction iPSCs, its transdifferentiation efficiency can reach 25%.In addition, induction iPSCs provided by the invention is divided into the method for beta Cell of islet, only needs, on selected suitable substratum and cultivation basis sequentially, to cultivate in an orderly manner to cell, simple to operate controlled, is easy to realize industrialization.
Accompanying drawing explanation
Fig. 1 is the differentiation-inducing front cellular form figure of iPSCs in the human skin source that the embodiment of the present invention provides;
Fig. 2 is the cellular form figure of the differentiation-inducing 4d of iPSCs in the human skin source that the embodiment of the present invention provides;
Fig. 3 is the cellular form figure of the differentiation-inducing 8d of iPSCs in the human skin source that the embodiment of the present invention provides;
Fig. 4 is the cellular form figure of the differentiation-inducing 13d of iPSCs in the human skin source that the embodiment of the present invention provides;
Fig. 5 is the cellular form figure of the differentiation-inducing 20d of iPSCs in the human skin source that the embodiment of the present invention provides.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiments provide a kind of method that iPSCs of induction is divided into beta Cell of islet, comprise the following steps,
S01. provide iPSCs, and use culture medium A to cultivate;
S02. until described iPSCs grow to more than 90% merge time, use collagenase IV to carry out digestion process, be separated into the little clump cells of iPSCs;
S03. little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells merges to 55-65%, described cell is cultivated in substratum B, obtain cell B, wherein, described substratum B is the DMEM/F12 substratum of BSA, 0.45-0.55 containing 0.18-0.22% × N2,0.45-0.55 × B27,95-105ng/mL activin A and 0.08-0.12 μM of wortmannin;
S04. described cell B is cultivated in culture medium C, obtain cell C;
S05. described cell C is cultivated in substratum D, obtain cell D;
S06. described cell D is cultivated in substratum E, obtain beta Cell of islet.
Concrete, in above-mentioned steps S01, described iPSCs a kind ofly has the of self-replication capacity and can be divided into the stem cell of multiple target function cell under certain condition, and it has abundance and does not relate to the advantages such as ethics problem.In the embodiment of the present invention, be induced to differentiate into the high transdifferentiation efficiency of beta Cell of islet in order to obtain iPSCs, described iPSCs is preferably the iPSCs in human skin source.Adopt the iPSCs in human skin source, described iPSCs can be made to be induced to differentiate into the transdifferentiation efficiency of beta Cell of islet up to 25%.
Obtain described iPSCs, need described iPSCs to cultivate.In order to obtain the iPSCs that cellular form is good, be applicable to conversion, the embodiment of the present invention preferably will be covered with the people ES cell of Matrigel Matrix without the culture medium A of feeder layer perfect medium as described iPSCs.As another preferred embodiment, the culture condition of described iPSCs in described culture medium A is: volumn concentration is the CO of 4.5-5.5%
2, 36-38 DEG C incubator in cultivate, be more preferably the CO that volumn concentration is 5%
2, cultivate in the incubator of 37 DEG C.
In above-mentioned steps S02, until described iPSCs grow to more than 90% merge time, use collagenase IV to carry out digestion process.As preferred embodiment, the temperature of described digestion process is 35-40 DEG C, and the concentration of described collagenase IV is 180-220U/mL, and digestion time is 3-5min.As further preferred embodiment, the temperature of described digestion process is 37 DEG C, and the concentration of described collagenase IV is 200U/mL, and digestion time is 4min.After this step, described iPSCs cellular segregation becomes the little clump cells of iPSCs, and undifferentiated cell clone gathers together through precipitation.
In above-mentioned steps S03, in the embodiment of the present invention, the selection of substratum is the key that induction iPSCs is divided into beta Cell of islet.In the embodiment of the present invention, little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, preferably when Growth of Cells to 60% merges, described cell is cultivated in substratum B.Further, in order to obtain good differentiation effect, described substratum B be preferably containing 0.2% BSA, 0.5 × N2,0.5 × B27,100ng/mL activin A and 0.1 μM wortmannin DMEM/F12 substratum.
As another preferred embodiment, under the condition of above-mentioned substratum B, the incubation time of described iPSCs is preferably 3-5d, more preferably 4d.
In above-mentioned steps S04, in order to effectively and promote the differentiation of described cell B in an orderly manner, as preferred embodiment, described culture medium C is the F12/IMDM substratum of ITS, the 0.45-0.55 × B27,1.8-2.2 μM RA, 18-22ng/ml FGF7 and 45-55ng/mL NOGGIN of BSA, 0.45-0.55% containing 0.45-0.55%.Further, described culture medium C be preferably containing 0.5% BSA, ITS, the 0.5 × B27 of 0.5%, the F12/IMDM substratum of 2 μMs of RA, 20ng/ml FGF7 and 50ng/mL NOGGIN.
As another preferred embodiment, under the condition of above-mentioned culture medium C, the incubation time of described cell B is preferably 3-5d, more preferably 4d.
In above-mentioned steps S05, in order to effectively and promote the differentiation of described cell C in an orderly manner, as preferred embodiment, described substratum D is the DMEM in high glucose substratum of ITS, the 0.8-1.2 × N2 and 45-55ng/mL EGF of BSA, 0.8-1.2% containing 0.45-0.55%.Further, described substratum D is the DMEM in high glucose substratum containing the BSA of 0.5%, ITS, the 1 × N2 of 1% and 50ng/mL EGF.
As another preferred embodiment, under the condition of above-mentioned substratum D, the incubation time of described cell C is preferably 4-6d, more preferably 5d.
In above-mentioned steps S06, in order to effectively and promote the differentiation of described cell D in an orderly manner, and then acquisition differentiation and maturation, and the cell that form, size are all similar with beta Cell of islet, as preferred embodiment, described substratum E is the DF12 substratum of ITS, 8-12ng/mL bFGF, 8-12mM nicotinamide, 45-55ng/mL Exendin-4 and the 8-12ng/mL BMP4 containing 0.8-1.2%.Further, described substratum E is the DF12 substratum of ITS, 10ng/mL bFGF, 10mM nicotinamide, 50ng/mL Exendin-4 and the 10ng/mL BMP4 containing 1%.
As another preferred embodiment, under the condition of above-mentioned substratum E, the incubation time of described cell D is preferably 6-8d, more preferably 7d.
As the optimum embodiment of the present invention, described induction iPSCs is divided into the method for beta Cell of islet, comprises the following steps:
The iPSCs that human skin is originated is provided, and uses the people ES cell being covered with Matrigel Matrix to cultivate without feeder layer perfect medium;
Until the iPSCs in described human skin source grow to more than 90% merge time, at 37 DEG C, carry out digestion process with the collagenase IV that concentration is 200U/mL, digestion 4min, described iPSCs are separated into the little clump cells of iPSCs;
Little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells to 60% merges, described cell is cultivated in the DMEM/F12 substratum containing 0.2%BSA, 0.5 × N2,0.5 × B27,100ng/mL activin A and 1 μM wortmannin, obtains cell B;
Described cell B is cultivated in the F12/IMDM substratum containing 0.5%BSA, 0.5%ITS, 0.5 × B27,2 μMs of RA, 20ng/ml FGF7 and 50ng/mL NOGGIN, obtains cell C;
Described cell C is cultivated in the DMEM in high glucose substratum containing 0.5%BSA, 1%ITS, 1 × N2 and 50ng/mL EGF, obtains cell D;
Described cell D is cultivated in containing the DF12 substratum of 1%ITS, 10ng/mL bFGF, 10mM nicotinamide, 50ng/mLExendin-4 and 10ng/mL BMP4, obtains beta Cell of islet.
The induction iPSCs that the embodiment of the present invention provides is divided into the method for beta Cell of islet, and the selection of substratum is the key that induction iPSCs is divided into beta Cell of islet.Each substratum that the embodiment of the present invention provides, its component and content thereof or concentration all have strict control.There is change arbitrarily in the composition of each substratum, all by affecting the differentiation of iPSCs to beta Cell of islet, can not obtain the differentiated result of embodiment of the present invention effect, even causing the failure broken up.
The induction iPSCs provided according to the embodiment of the present invention is divided into the method for beta Cell of islet, cell cultures is carried out according to specific order by selecting multiple specific substratum, can within the time of 20 days, iPSCs is induced to differentiate into beta Cell of islet, and transdifferentiation efficiency can reach 25% efficiently and directionally.In addition, method described in the embodiment of the present invention, only needs, on selected suitable substratum and cultivation basis sequentially, to cultivate in an orderly manner to cell, simple to operate controlled, is easy to realize industrialization.
Be described below in conjunction with specific embodiment.
Induce iPSCs to be divided into a method for beta Cell of islet, comprise the following steps:
S11., the iPSCs that human skin is originated is provided, and uses the people ES cell being covered with Matrigel Matrix to cultivate without feeder layer perfect medium;
S12. until the iPSCs in described human skin source grow to more than 90% merge time, at 37 DEG C, carry out digestion process with the collagenase IV that concentration is 200U/mL, digestion 4min, described iPSCs are separated into the little clump cells of iPSCs;
S13. little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells to 60% merges, described cell is carried out cultivation 4d in the DMEM/F12 substratum containing 0.2%BSA, 0.5 × N2,0.5 × B27,100ng/mL activin A and 1 μM wortmannin, obtains cell B;
S14. described cell B is carried out cultivation 4d in the F12/IMDM substratum containing 0.5%BSA, 0.5%ITS, 0.5 × B27,2 μMs of RA, 20ng/mlFGF7 and 50ng/mL NOGGIN, obtain cell C;
S15. described cell C is carried out cultivation 5d in the DMEM in high glucose substratum containing 0.5%BSA, 1%ITS, 1 × N2 and 50ng/mL EGF, obtain cell D;
S16. described cell D is carried out cultivation 7d in containing the DF12 substratum of 1%ITS, 10ng/mL bFGF, 10mM nicotinamide, 50ng/mL Exendin-4 and 10ng/mL BMP4, obtain beta Cell of islet.
Before the differentiation provide embodiment of the present invention step S11, described iPSCs carries out morphological observation under 20 times of inverted microscopes, as shown in Figure 1.As seen from the figure, described iPSCs Growth of Cells is good, and clone is fine and close.
Cell after the described iPSCs provided by embodiment of the present invention step S13 cultivates 4d in described substratum B carries out morphological observation under 20 times of inverted microscopes, as shown in Figure 2.As seen from the figure, cellular form changes, and starts to break up to pancreas direction.
Cell after the described cell B that embodiment of the present invention step S14 provides cultivates 4d in described substratum carries out morphological observation under 20 times of inverted microscopes, as shown in Figure 3.As seen from the figure, cellular form changes, and cell breaks up to progenitor cell direction.
Cell after the described cell C that embodiment of the present invention step S15 provides cultivates 5d in described substratum carries out morphological observation under 4 times of inverted microscopes, as shown in Figure 4.As seen from the figure, there is obviously amplification in progenitor cell.
Cell after the described cell D that embodiment of the present invention step S16 provides cultivates 7d in described substratum carries out morphological observation under 4 times of inverted microscopes, as shown in Figure 5.As seen from the figure, ancestor cell differentiates is ripe, all similar beta Cell of islet of form, size.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. induce iPSCs to be divided into a method for beta Cell of islet, comprise the following steps,
IPSCs is provided, and uses culture medium A to cultivate;
Until described iPSCs grow to more than 90% merge time, use collagenase IV to carry out digestion process, be separated into the little clump cells of iPSCs;
Little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells merges to 55-65%, described cell is cultivated in substratum B, obtain cell B, wherein, described substratum B is the DMEM/F12 substratum of BSA, 0.45-0.55 containing 0.18-0.22% × N2,0.45-0.55 × B27,95-105ng/mL activin A and 0.08-0.12 μM of wortmannin;
Described cell B is cultivated in culture medium C, obtains cell C;
Described cell C is cultivated in substratum D, obtains cell D;
Described cell D is cultivated in substratum E, obtains beta Cell of islet.
2. induction iPSCs as claimed in claim 1 is divided into the method for beta Cell of islet, and it is characterized in that, described iPSCs is the iPSCs of people's skin-derived.
3. induction iPSCs as claimed in claim 1 is divided into the method for beta Cell of islet, it is characterized in that, described culture medium A be covered with Matrigel Matrix people ES cell without feeder layer perfect medium.
4. the induction iPSCs as described in as arbitrary in claim 1-3 is divided into the method for beta Cell of islet, it is characterized in that, described culture medium C is the F12/IMDM substratum of ITS, the 0.45-0.55 × B27,1.8-2.2 μM RA, 18-22ng/ml FGF7 and 45-55ng/mL NOGGIN of BSA, 0.45-0.55% containing 0.45-0.55%.
5. the induction iPSCs as described in as arbitrary in claim 1-3 is divided into the method for beta Cell of islet, it is characterized in that, described substratum D is the DMEM in high glucose substratum of ITS, the 0.8-1.2 × N2 and 45-55ng/mL EGF of BSA, 0.8-1.2% containing 0.45-0.55%.
6. the induction iPSCs as described in as arbitrary in claim 1-3 is divided into the method for beta Cell of islet, it is characterized in that, described substratum E is the DF12 substratum of ITS, 8-12ng/mL bFGF, 8-12mMnicotinamide, 45-55ng/mL Exendin-4 and the 8-12ng/mL BMP4 containing 0.8-1.2%.
7. the induction iPSCs as described in as arbitrary in claim 1-3 is divided into the method for beta Cell of islet, it is characterized in that, described use collagenase IV carries out in the step of digestion process, the temperature of described digestion process is 35-40 DEG C, the concentration of described collagenase IV is 180-220U/mL, and digestion time is 3-5min.
8. the induction iPSCs as described in as arbitrary in claim 1-3 is divided into the method for beta Cell of islet, it is characterized in that, comprises the following steps,
The iPSCs that human skin is originated is provided, and uses the people ES cell being covered with Matrigel Matrix to cultivate without feeder layer perfect medium;
Until the iPSCs in described human skin source grow to more than 90% merge time, at 37 DEG C, carry out digestion process with the collagenase IV that concentration is 200U/mL, digestion 4min, described iPSCs are separated into the little clump cells of iPSCs;
Little for described iPSCs clump cells is inoculated on the substratum being covered with MATRIGEL MATRIX and cultivates, when Growth of Cells to 60% merges, described cell is cultivated in the DMEM/F12 substratum containing 0.2%BSA, 0.5 × N2,0.5 × B27,100ng/mL activin A and 1 μM wortmannin, obtains cell B;
Described cell B is cultivated in the F12/IMDM substratum containing 0.5%BSA, 0.5%ITS, 0.5 × B27,2 μMs of RA, 20ng/ml FGF7 and 50ng/mL NOGGIN, obtains cell C;
Described cell C is cultivated in the DMEM in high glucose substratum containing 0.5%BSA, 1%ITS, 1 × N2 and 50ng/mL EGF, obtains cell D;
Described cell D is cultivated in containing the DF12 substratum of 1%ITS, 10ng/mL bFGF, 10mM nicotinamide, 50ng/mLExendin-4 and 10ng/mL BMP4, obtains beta Cell of islet.
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Application publication date: 20150916 |