CN102732479A - Preparation method for thymic epithelial precursor cells and special medium therefor - Google Patents
Preparation method for thymic epithelial precursor cells and special medium therefor Download PDFInfo
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Images
Abstract
The invention discloses a preparation method for thymic epithelial precursor cells and a special medium therefor. The thymic epithelial precursor cells provided in the invention are expression obtained through differentiation of human embryonic stem cells or induced pluripotent stem cells, are thymic epithelial precursor cells of Epcam, Cytokeratin 5 and Cytokeratin 8, and have proliferation capacity and the potential of differentiation into medullary thymic epithelial cells and cortical thymic epithelial cells and/or promotion of maturation of T cells. According to results of experiments in the invention, a staged induction system for differentiation of human embryonic stem cells into thymic epithelial precursor cells is established for the first time in the invention by using the method of in-vitro induction and simulation of the process of thymus development in vivo.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of preparation method and special culture media thereof of thymus epithelial precursor cell.
Background technology
Thymus gland is the immune organ of inducing T cell differentiation and maturation, has vital role for keeping of the normal function of human immune system.Can many diseases relevant with immunity system all induce the formation of normal T cell closely related with thymus gland.For example, the DiGeorge syndromes promptly is because the immunodeficient disease that congenital thymic aplasia causes.Can treat this disease effectively through the allosome thymus transplantation, in the existing clinical application of the U.S..On the other hand, thymus gland also has the critical function of inducing immune tolerance, and this is extremely important for the allosome organ transplantation and based on the treatment of cell replacement.Allosome organ transplantation at present needs to take immunosuppressor after surgery for a long time mostly, also brings a series of problems to comprise thus: (one) long term inhibition body's immunity can greatly increase the risk of lethal infection and tumour generation; (2) most of immunosuppressor itself have the intensive toxic side effect; (3) most immunosuppressor prices are very expensive, and life-time service is too high as far as patient's cost.Induce acceptor to allosome or xenotransplantation deposits yields immunological tolerance through thymus transplantation, can effectively break away from a series of problems that the life-time service immunosuppressor is brought, have high clinical value.In addition, after human pubescence with advancing age thymus gland senesce, replaced by fatty tissue gradually, following people older and that immunological competence is low also can benefit from thymus transplantation.Thymus transplantation has broad clinical application prospect in a word.But thymus gland itself is as a kind of organ, and its source receives great restriction, how to obtain a large amount of thymic tissues and is used for clinical treatment, is the subject matter of carrying out the relevant clinical treatment of thymus gland at present.
Human embryo stem cell can be to the differentiation potential of three germinal layer all cells of human body type and in external long-term a large amount of amplifications, for the source problem that comes that solves the thymus gland organ provides a kind of possible solution owing to having.Therefore, induce differentiation to obtain the thymic epithelial cells, have crucial clinical value through human embryo stem cell.But the embryonic stem cell of still having no talent is at present induced the report of differentiation to thymus gland.Under the sophisticated relatively situation of thymus transplantation clinical protocol, effectively inducing human embryo stem cell is the main bottleneck that thymus transplantation is applied to numerous clinical applications to the foundation of the differentiated system of thymus gland differentiation.
Realize the differentiation of inducing of myeloid-lymphoid stem cell in dissimilar cytodifferentiation, all being able to prove a kind of very successful strategy through the development pathway of specific cells in the analogue body or organ.Growth course has accumulated some clues in the body of thymus gland in the research that mice embryonic is grown.When mice embryonic was grown the E11.5 days left and right sides, primordium of thymus produced from endoblastic third pharyngeal pouch position.At approximately about E12 days; The primordium of thymus specialization becomes a group epithelium precursor cell; This group precursor cell can further be differentiated to form the two kinds of cell types (cortex epithelial cell and medullary epithelial cell) that constitute thymus gland future; Discover that only single thymus epithelial precursor cell just has the ability that can rebuild whole thymus gland.About E13.5 days, the structure of thymus gland basically forms.The also existing research to a certain degree of key gene in these a series of growth courses and signal path.For example, Foxn1 is the key gene of thymus development.In mouse, knock out the Foxn1 gene and can cause the disappearance fully of thymus gland.But more the thymus development process of details, the particularly correlative study of the growth course of people's thymus gland are still deficient.Therefore, set up human embryo stem cell, also have very important meaning in vitro study people thymus development process to thymocyte induced differentiation system.
Summary of the invention
An object of the present invention is to provide a kind of thymus epithelial precursor cell.
Thymus epithelial precursor cell provided by the invention; Be the thymus epithelial precursor cell of the expression, epithelial cell adhesion molecule, cytokeratin 5 and the cytokeratin 8 that are obtained by the differentiation of human embryo stem cell or inductive pluripotent stem cell, it has multiplication capacity and to the potential of thymic medulla epithelial cell with differentiation of thymic cortex epithelial cell and/or promotion T cell maturation.
Said human embryo stem cell is can be from the human embryonic stem cell of commercial sources acquisition.
Said can be following any clone from the human embryonic stem cell that commercial sources obtains: BG01, BG02, BG03, BG04, SA01, SA02, SA03; ES01, ES02, ES03, ES04, ES05, ES06, TE03; TE32, TE33, TE04, TE06, TE62, TE07, TE72; UC01, UC06, WA01, WA07, WA09, WA13 and WA14; The said numbering that is numbered NIH.
Another object of the present invention provides a kind of substratum by human embryo stem cell or the said thymus epithelial precursor cell of inductive pluripotent stem cell differentiation preparation, forms by cell culture fluid I, cell culture fluid II and cell culture fluid III,
Said cell culture fluid I prepares according to following method: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, activin A and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol and activin A is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (80-120) ng;
Said cell culture fluid II prepares according to following method: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid, IWP2 and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid and IWP2 is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (0.8-1.2) nmol: (2.3-2.8) nmol;
IWP2 is a kind of Wnt Inhibitor.
Said cell culture fluid III obtains according to following method preparation: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4, wnt3a and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4 and wnt3a is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (80-120) ng: (80-120) ng.
Among the said cell culture fluid I, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol and activin A is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng;
Among the said cell culture fluid II, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid and IWP2 is 1ml: 0.01ml: 0.01ml: 0.001ml: 1nmol: 2.5nmol;
Among the said cell culture fluid III, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4 and wnt3a is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng: 100ng.
The basic medium of said cultivation mammalian cell is X-VIVO 10 substratum.
The 3rd purpose of the present invention provides the method for the said thymus epithelial precursor cell of the said medium preparation of a kind of usefulness, comprises the steps:
1) with cultivating on human embryo stem cell or the cell culture fluid I of inductive pluripotent stem cell in said substratum;
2) cultivate on the cell culture fluid II of cell in said substratum that step 1) obtains;
3) cultivate on the cell culture fluid III of cell in said substratum that step 2) obtains, obtain the thymus epithelial precursor cell.
In the step 1), said incubation time is 2.5-3.5 days, and said incubation time was specially 3 days;
Step 2) in, said incubation time is 2.5-4 days, and said incubation time was specially 4 days;
In the step 3), said incubation time is 4.5-5.5 days, and said incubation time was specially 5 days.
Said human embryo stem cell is can be from the human embryonic stem cell of commercial sources acquisition.
Said can be following any clone from the human embryonic stem cell that commercial sources obtains: BG01, BG02, BG03, BG04, SA01, SA02, SA03; ES01, ES02, ES03, ES04, ES05, ES06, TE03; TE32, TE33, TE04, TE06, TE62, TE07, TE72; UC01, UC06, WA01, WA07, WA09, WA13 and WA14; The said numbering that is numbered NIH;
Described cell, described method or described substratum are at preparation thymic medulla epithelial cell and thymic cortex epithelial cell and/or promote that the application in the T cell maturation also is the scope that the present invention protects.
Said T cell is the CD4+T cell.
Wherein, said human embryo stem cell is originated and is seen table 1.
Table 1. can be from the human embryonic stem cell of commercial sources acquisition
Of the present invention experiment showed, in the present invention through the method for external evoked analogue body internal thymus growth course, set up human embryo stem cell first and induced system stage by stage to the differentiation of thymus epithelial precursor cell.Originally inducing in the differentiated system, human embryo stem cell can efficiently break up along the growth path of entoderm-third pharyngeal pouch-thymus epithelial precursor.Can the differentiation different steps detect entoderm successively, third pharyngeal pouch, the thymus epithelial genes involved on the mRNA level with protein level on expression.What is more important; With inductive people thymus epithelial precursor cell be transplanted in the nude mouse of athymism after the human embryonic fibroblast mixes; Can induce because of producing the T cell in the nude mouse that causes lacking mature T cells from the body athymism; Proved that people's thymus epithelial precursor cell of inducing acquisition can be formed with the thymus structure of function in vivo, had the ability that inducing T cell forms.The external system of inducing of breaking up to the thymus epithelial precursor cell of inducing stage by stage of human embryo stem cell that the present invention sets up has very important meaning in vitro study people thymus development process.What is more important; Induce people's thymus epithelial precursor cell of acquisition to have the ability of the thymus gland that is formed with function; This provides the potential organ origin for the application that thymus transplantation is relevant clinically, thereby can greatly promote the application of thymus transplantation aspect treatment autoimmune disorders and allosome organ transplantation.
Differentiation scheme provided by the invention is in the in-vitro simulated process of people's thymus development in the body, has very important significance for the process of research people thymus development.This differentiation scheme can be used for obtaining the thymus epithelial precursor cell in a large amount of human embryo stem cells source, and these precursor cells can be used for extensive clinical application and the research based on thymus transplantation.
Description of drawings
Fig. 1 is genetic expression in the RT-PCR performance analysis atomization
Fig. 2 detects the protein expression situation of three differential period typical case genes for immuno-fluorescent antibody technique
Fig. 3 for flow cytometer showed detect second differential period and the 3rd differential period the presentation markup gene cell proportion
Fig. 4 detects the ratio of H9 clone differentiation thymus epithelial precursor cell in latter stage for flow cytometer showed
Fig. 5 analyzes the thymus epithelial precursor cell graft structure in the human embryo stem cell source of transplanting in the body for immuno-fluorescent antibody technique
Whether the thymus epithelial precursor cell that Fig. 6 originates for the human embryo stem cell of transplanting in the streaming method detection bodies supports the formation of T cell
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The prescription of PBS: take by weighing 40 gram sodium-chlor; 7.16 the gram disodium hydrogen phosphate, 1 gram Repone K and 1 gram potassium primary phosphate are dissolved in 500 milliliter of 18,000,000 water, are prepared into 10XPBS as liquid storage; With 10 times of 18,000,000 water dilutions, become the 1XPBS that can directly use during use.
The preparation of embodiment 1, thymus epithelial precursor cell and evaluation
One, inducing human embryo stem cell was identified to foundation and each stage of thymus epithelial precursor cell directed differentiation system
(1) foundation of differentiated system
The result of study of at first growing according to mouse thymus was divided into for three steps with the whole atomization of inducing: (1) breaks up entoderm to the embryo from embryonic stem cell; (2) break up to the entoderm third pharyngeal pouch from entoderm in the embryo; (3) break up to the thymus epithelial precursor cell from the entoderm third pharyngeal pouch.Subsequently, confirmed each differential period is played a crucial role and the gene of mark effect: fs Sox17 and Foxa2, subordinate phase Hoxa3 and Tbx1, phase III K5 and K8.To these expression of gene, the signal path relevant to thymus development screens, and finally confirmed following differentiation scheme:
(1) stand density is reached (1X10
4) human embryonic stem cell H1 (Wisconsin Alumni Research Foundation WA01) presses inoculum density (1X10 with dispase digestion back
4) be inoculated in Matrigel and encapsulate on the petridish of (concentration 2%), nutrient solution personnel selection ES nutrient solution was cultivated 24 hours under temperature is 37 ℃, 5% carbon dioxide conditions.
(2) cell that step (1) is obtained is used nutrient solution I instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 3 days.
(3) cell that step (2) is obtained digests the back by 4X10 with 0.25%trypsin
5/ cm
2Density be inoculated in the petridish that Matrigel (concentration 2%) encapsulates.Under temperature is 37 ℃, 5% carbon dioxide conditions, continue to cultivate 2 days with nutrient solution I.
(4) cell that step (3) is obtained is used nutrient solution II instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 4 days.
(5) cell that step (4) is obtained is used nutrient solution III instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 5 days.
Said cell culture fluid I obtains according to following method preparation: with X-VIVO 10 substratum (Lonza company; Catalog number is 04-380Q), Glutamax (NSC 334200 dipeptides, Invitrogen Company products catalog number (Cat.No.) is 35050), NEAA (Invitrogen company; Catalog number is 11140), beta-ME (2 mercapto ethanol, Invitrogen company; Catalog number is 21985-023) and activin A (activin A, Peprotech; Catalog number: 120-14) mix, obtain cell culture fluid I, wherein the proportioning of X-VIVO 10 substratum, Glutamax, NEAA, beta-ME and activin A is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng, and the pH value is 7.0.
Said cell culture fluid II obtains according to following method preparation: with X-VIVO 10 substratum, Glutamax (NSC 334200 dipeptides, Invitrogen Company products catalog number (Cat.No.) is 35050), NEAA (Invitrogen company; Catalog number is 11140), beta-ME (2 mercapto ethanol, Invitrogen company; Catalog number is 21985-023), RA (vitamin A acid, Sigma company; Catalog number: R2625) and IWP2 (Merck company; Catalog number 681671) mix, obtain cell culture fluid II, wherein the proportioning of X-VIVO 10 substratum, Glutamax, NEAA, beta-ME, RA and IWP2 is 1ml: 0.01ml: 0.01ml: 0.001ml: 1nmol: 2.5nmol, and the pH value is 7.0.
Said cell culture fluid I I I obtains according to following method preparation: with X-VIVO 10 substratum, Glutamax (NSC 334200 dipeptides, Invitrogen Company products catalog number (Cat.No.) is 35050), NEAA (Invitrogen company; Catalog number is 11140), beta-ME (2 mercapto ethanol, Invitrogen company; Catalog number is 21985-023), BMP4 (bone morphogenetic protein 4, R&D company; Catalog number: 314-BP) and wnt3a (R&D company; Catalog number is 1324-WN) mix; Obtain cell culture fluid III; Wherein the proportioning of X-VIVO 10 substratum, Glutamax, NEAA, beta-ME, BMP4 and wnt3a is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng: 100ng, and the pH value is 7.0.
(2) the external evaluation of each differential period
1. adopt different steps expression conditions in the whole vitro differentiation process of RT-PCR detection of dynamic.
Induce the division (entoderm was induced 0-5 days, and third pharyngeal pouch was induced 6-9 days, and the thymus epithelial precursor cell was induced 10-14 days) of system according to three-step approach, respectively in atomization 0; 3,5,9; Carry out RT-PCR in 14 days and detect human embryo stem cell specific gene Oct4 in the differentiated system, entoderm specific gene CXCR4, third pharyngeal pouch specific gene Eya1, Six1; Thymus epithelial specific gene AIRE, Pax9, the expression of Foxn1, the required primer of each gene is as shown in table 1.
Table 1 is for detecting primer sequence
The result is as shown in Figure 1, and D0, D3, D5, D9, D14 represent the 0th, 3,5,9,14 day respectively, and along with the carrying out of atomization, giving expression to now of Oct4 transferred.CXCR4 did not express at the 0th day, and tangible rise was arranged at the 3rd, 5 day, and significantly downward modulation occurred at the 9th day.Eya1 and Dix1 all have than strongly expressed at human embryo stem cell, but along with its expression of carrying out of differentiation can weaken gradually, expression is reduced to minimum in the latter stage of entoderm differentiation.And after inducing through the third pharyngeal pouch stage, the expression of Eya1 and Dix1 significantly strengthens again.Pax9 also has than strongly expressed in human embryo stem cell, but along with its expression of carrying out of differentiation can weaken to disappearance gradually, induces the back it is expressed just and occurs again having passed through the thymus epithelial precursor cell stage.AIRE, Foxn1 then only just express after inducing through the thymus epithelial precursor cell stage as thymic epithelial cells's specific expression gene.
These presentation of results, on the mRNA level, process that the three-step approach differentiated system is grown at in-vitro simulated body internal thymus has experienced stage of embryonic stem cell, entoderm, third pharyngeal pouch, thymus epithelial successively.
2. adopt immuno-fluorescent antibody technique to detect the protein expression situation of three differential period typical case genes.
Antibody information:
The antibody that detects SOX17 is the monoclonal antibody in mouse source, and available from P&ABiotech, catalog number is A07074-2F9.
The antibody that detects Foxa2 is the how anti-of sheep source, and available from R&D, catalog number is AF2400.
The antibody that detects Hoxa3 is the how anti-of rabbit source, and available from Santa cruz, catalog number is sc-28598.
The antibody that detects Tbx1 is the how anti-of rabbit source, and available from abcam, catalog number is ab18530.
The antibody that detects Cytokeratin 5 is the how anti-of rabbit source, and available from abcam, catalog number is ab24647.
The antibody that detects Cytokeratin 8 is the monoclonal antibody in mouse source, and available from abcam, catalog number is ab9023.
Two anti-Donkey-anti-Mouse-488 are available from Jackson ImmunoResearch, and catalog number is 715-485-151.
Two anti-Donkey-anti-Mouse-549 are available from Jackson ImmunoResearch, and catalog number is 715-505-151.
Two anti-Donkey-anti-Goat-549 are available from Jackson ImmunoResearch, and catalog number is 705-505-147.
Two anti-Donkey-anti-Rabbit-488, available from Jackson ImmunoResearch, catalog number is 711-485-152.
The immuno-fluorescent antibody technique detection has been carried out to the specific genes of three etap in the thymus development process in atomization the 3rd, 5,9,14 days.
Specific as follows:
1) reagent is prepared
Liquid is changed in sealing thoroughly: take by weighing 0.5g BSA and be dissolved among the 45ml PBS, and add 55 μ l Triton X-100 and 5ml sealing and use notmal horse sera, mixing is put in 4 and spends preservation;
Antibody diluent: take by weighing 0.1g BSA, be dissolved among the PBS of 100ml, promptly be made into 0.1% BSA solution;
The DAPI working fluid: DAPI is diluted among the PBS, and making its final concentration is 1 μ g/ml.
2) operation steps
1) takes out cell, discard substratum, wash one time with the PBS of preheating;
2) Paraformaldehyde 96 of adding 4%, room temperature (25 degree) was placed 20 minutes;
3) give a baby a bath on the third day after its birth time each 3 minutes with PBS;
4) in cell, add sealing and change liquid thoroughly, room temperature was placed 45 minutes;
5) give a baby a bath on the third day after its birth time each 3 minutes with PBS;
6) add anti-(in the fs of differentiation, promptly a differentiation 3 days and the 5th day: Sox17 (dilution in 1: 200) and Foxa2 (dilution in 1: 200); The subordinate phase of differentiation was promptly broken up the 9th day: Hoxa3 (dilution in 1: 50) or Tbx1 (dilution in 1: 200; The phase III of differentiation promptly broke up the 14th day: Cytokeratin 5 (dilution in 1: 1000) and Cytokeratin8 (dilution in 1: 200) are diluted in the antibody diluent), 4 degree are placed and are spent the night;
7) give a baby a bath on the third day after its birth time each 5 minutes with PBS;
8) add that corresponding two anti-(fs two is anti-to be: Donkey-anti-Mouse-488 and Donkey antiGoat-549; Subordinate phase two resists: Donkey-anti-rabbit-549; Phase III two resists: Donkey-anti-rabbit-488 and Donkey-anti-mouse-549 are 1: 200 and are diluted in the antibody diluent), room temperature (25 degree) was placed 1 hour;
9) wash once 3 minutes with PBS;
10) add the DAPI working fluid, room temperature was placed 5 minutes;
11) give a baby a bath on the third day after its birth time each 3 minutes with PBS;
12) add PBS, sample is observed under fluorescent microscope and is taken pictures.
(Fig. 2 A is the result of differentiation detection in the 3rd day Sox17, Foxa2, and Fig. 2 B is for breaking up the result who detected Sox17, Foxa2 on the 5th day, and Fig. 2 C is for breaking up the result who detected Hoxa3 on the 9th day for result such as Fig. 2; Fig. 2 D detected the result of Tbx1 on the 9th day for differentiation; Fig. 2 E detected the result of K5 and K8 on the 14th day for differentiation) shown in, in atomization the 3rd day can detect the higher Sox17 of ratio in the differentiated system; The single positive cell of Foxa2 and a certain proportion of pair of positive cell are shown in Fig. 2 A.Sox17 and Foxa2 are endoblastic specific gene.In atomization the 5th day, Sox17, the ratio of the two positive cells of Foxa2 has further lifting, shown in Fig. 2 B.In atomization the 9th day can detect the cell of a certain proportion of expression third pharyngeal pouch specific gene Tbx1, shown in Fig. 2 D, also can detect the cell of expressing another specific gene of third pharyngeal pouch Hoxa3 simultaneously, shown in Fig. 2 C.In addition, just can detect the cell of expressing Cytokeration 5 (K5) and Cytokeration 8 (K8) simultaneously at the 9th day, shown in Fig. 2 E.Expressing Cytokeration 5 simultaneously is notable features of thymus epithelial precursor cell with Cytokeration 8; Cytokeratin 5 in growth course, and 8 pairs of male thymus epithelials of Cytokeratin precursor cell can be differentiated to form Cytokeratin 5 single male thymic medulla epithelial cells and Cytokeratin 8 single male thymic cortex epithelial cells.
Therefore, on protein level, further prove conclusively the process that the three-step approach differentiated system is grown at in-vitro simulated body internal thymus, experienced the stage of embryonic stem cell, entoderm, third pharyngeal pouch, thymus epithelial successively.
3. adopt the method for flow cytometer showed detect second differential period and the 3rd differential period the presentation markup gene cell proportion.
Antibody information:
The antibody that detects Hoxa3 is the how anti-of rabbit source, and available from Santa cruz, catalog number is sc-28598.
The antibody that detects Tbx1 is the how anti-of rabbit source, and available from abcam, catalog number is ab18530.
The antibody that detects Cytokeratin 5 is the how anti-of rabbit source, and available from abcam, catalog number is ab24647.
The antibody that detects Cytokeratin 8 is the monoclonal antibody in mouse source, and available from abcam, catalog number is ab9023.
The antibody that detects Anti-Epcam-PE is the straight mark monoclonal antibody in mouse source, and available from BD, catalog number is 347198.
Two anti-Donkey-anti-Mouse-649 are available from Jackson ImmunoResearch, and catalog number is 715-495-151
Two anti-Donkey-anti-Goat-488 are available from Jackson ImmunoResearch, and catalog number is 705-485-147
Two anti-Donkey-anti-Rabbit-649 are available from Jackson ImmunoResearch, and catalog number is 711-495-152
Two anti-Donkey-anti-Rabbit-488, available from Jackson ImmunoResearch, catalog number is 711-485-152
The cell of collect differentiated system the 9th day and the 14th day is purchased cell fixation/penetrating test kit (catalog number the is 554714) specification sheets that comes according to BD Biosciences and is prepared analytic sample, analyzes with flow sorter.
Flow cytometer showed dyeing experimental procedure:
1) need carry out the cell of flow cytometer showed in the particular point in time collection, (the Invitrogen company of the pancreatin with 0.25%; Catalog number is: 25200) digestion, and digestion time is about 5 minutes;
2) with the nutrient solution termination reaction that contains serum;
3) blow and beat cell repeatedly with the rifle head, make agglomerate dispelled into individual cells;
4) centrifugal, 1200 change 5 minutes;
5) abandon supernatant after, wash twice with PBS;
6) with 200ul BD Fixation/Permeabilization (BD company, catalog number is: 51-2090KZ) re-suspended cell, 4 the degree hatched 20 minutes;
7) with 1XBD Perm/Wash Buffer (BD company, catalog number is: 51-2090KZ) washing is 3 times, and 1200 change, centrifugal 5 minutes;
8) add anti-(in the fs of differentiation, promptly a differentiation 3 days and the 5th day: Sox17 (dilution in 1: 200) and Foxa2 (dilution in 1: 200); The subordinate phase of differentiation was promptly broken up the 9th day: Hoxa3 (dilution in 1: 50) or Tbx1 (dilution in 1: 200; The phase III of differentiation promptly broke up the 14th day: Cytokeratin 5 (dilution in 1: 1000) and Cytokeratin8 (dilution in 1: 200) are dissolved among the 1XBD Perm/Wash Buffer), 4 degree were placed 30 minutes;
9) with 1XBD Perm/Wash Buffer washing 3 times, 1200 change centrifugal 5 minutes;
10) add that two anti-(fs two is anti-to be: Donkey-anti-Mouse-649 and Donkey anti Goat-488; Subordinate phase two resists: Donkey-anti-rabbit-649 and Anti-EpCAM-PE; Phase III two resists: Donkey-anti-rabbit-488 and Donkey-anti-mouse-649; Be dissolved among the 1XBD Perm/Wash Buffer; Removing Anti-EPCAM-PE is that working concentration is 0.6ug/ml, and all the other are dilution in 1: 200), 4 degree were placed 30 minutes;
11) with 1XBD Perm/Wash Buffer washing 3 times, 1200 change centrifugal 5 minutes;
12) add the PBS that 200ul contains 2% Paraformaldehyde 96, re-suspended cell;
13) flow type analyzer detects.
Anti-not add one; Only add of the contrast of corresponding two anti-cells as fs and phase III; The contrast of subordinate phase is that Donkey-anti-rabbit-649 and PE-anti-human IgG (available from Biolegend, catalog number 409303, working concentration is 2.5ug/ml) are contrast.
(Fig. 3 A detected the result of Tbx1 and Epcam on the 9th day for differentiation for result such as Fig. 3; Fig. 3 B detected the result of Hoxa3 and Epcam on the 9th day for differentiation; Fig. 3 C detected the result of K5 and K8 on the 14th day for differentiation) shown in, the 9th day about cell of about 70% of differentiated system expressed Tbx1, Epcam simultaneously, shown in Fig. 3 A; About cell of about 70% is expressed Hoxa3, Epcam simultaneously, shown in Fig. 3 B.Tbx1 and Hoxa3 are the important marker gene of third pharyngeal pouch, and the gene knockout experiment shows that the third pharyngeal pouch growth of Tbx1 knock-out mice and Hoxa3 knock-out mice is all undesired.Epcam is epithelial surface markers.These presentation of results can realize that in the differentiation subordinate phase third pharyngeal pouch is induced efficiently.Shown in Fig. 3 C, the 14th day about cell of about 25% of differentiated system expressed Cytokeratin 5 and Cytokeratin 8 simultaneously, explain breaking up the phase III and can realize that also the thymus epithelial precursor cell of greater efficiency induces.
Adopt aforesaid method; Except H1 clone; Also in H9 clone (available from Wisconsin Alumni Research Foundation; Catalog number is WA09) go up repetition the three-step approach on H1 clone, set up induce the differentiation scheme, adopt the method for flow cytometer showed, be contrast to add isotype IgG one anti-.The result is as shown in Figure 4, the final formation that also can detect Cytokeratin 5,8 pairs of male thymus epithelials of Cytokeratin precursor cell at the 14th day, and its ratio is about about 30%.
This this three-step approach of presentation of results induces the differentiation scheme to be applicable to other people embryonic stem cell line that H1 is outer.
The functional evaluation of the thymus epithelial precursor cell in the human embryo stem cell source of transplanting in embodiment 2, the body
1, immuno-fluorescent antibody technique is analyzed the thymus epithelial precursor cell graft structure in the human embryo stem cell source of transplanting in the body
Antibody information:
The antibody that detects Cytokeratin 5 is the how anti-of rabbit source, and available from abcam, catalog number is ab24647.
The antibody that detects Cytokeratin 8 is the monoclonal antibody in mouse source, and available from abcam, catalog number is ab9023.
With the kidney packing place that is transplanted to NOD/SCID mouse (Beijing China Fukang biotech inc) behind the 14th day the cell harvesting of differentiated system, immunohistochemical methods detects the expression of thymus epithelial marker gene Cytokeratin 5 and Cytokeratin 8 in the graft after one month.
Immunohistochemical detection method is specific as follows:
1) reagent is prepared
Liquid is changed in sealing thoroughly: take by weighing 0.5g BSA and be dissolved among the 45ml PBS; And add 55 μ l polyoxyethylene octyl phenyl ethers and 5ml sealing with normal donkey serum (Jackson ImmunoResearch company; Catalog number is 017-000-121), mixing is put in 4 degree and preserves;
Antibody diluent: take by weighing 0.1g BSA, be dissolved among the PBS of 100ml, promptly be made into 0.1% BSA solution;
The DAPI working fluid: DAPI is diluted among the PBS, and making its final concentration is 1 μ g/ml.
Buffering glycerine: the USP Kosher of 1ml is dissolved among the 1ml PBS, mixes.
2) operation steps
1. frozen section paster, room temperature was placed 30 minutes;
2. with immunohistochemical methods pen (company of middle China fir Golden Bridge; Catalog number is: ZLI-9305) around section, draw circle, room temperature was placed 10 minutes;
3. cold acetone (being placed on-20 ℃ of refrigerators in advance) soaks slide, and-20 degree were placed 10 minutes;
4.PBS washing, 5 minutes/inferior X3 time;
5. change fluid-tight thoroughly with about 200ul sealing and close, carry out in the wet box, room temperature was placed 1 hour;
6. add one anti-(being dissolved in antibody diluent): rabbit anti-cell Keratin sulfate 5 (concentration is 1:: 1000, Abcam company, catalog number is: ab24647) (concentration is 1: 500 with mouse-anti cytokeratin 8; Abcam company; Catalog number is: ab9023), carry out in the wet box, 4 spend night;
7.PBS washing, 5 minutes/inferior X3 time
8. add two anti-(being dissolved among the PBS): (concentration is donkey anti-rabbit-549: 1: 200; Jackson ImmunoResearch company, catalog number is 711-505-152) and donkey anti-mouse-488 (concentration is: 1: 200; Jackson ImmunoResearch company, catalog number is 715-485-151) to carry out in the wet box, room temperature was placed 1 hour
9.PBS wash 5 minutes/inferior X3 time
10.DAPI working fluid dyeing, room temperature 5 minutes
11.PBS wash 5 minutes/inferior X3 time
12. buffering glycerine mounting, every sheet is used 20ul
13. under fluorescent microscope, observe and the one-tenth phase.
Result such as Fig. 5 (detecting the result of K5 and K8 behind Fig. 5 A and Fig. 5 B thymus epithelial precursor cell for transplanting human embryo stem cell source on the 30th day) show; In graft; Can detect Cytokeratin 5; The two positive cells (shown in Fig. 5 B) of Cytokeratin8 also can detect the cell (shown in Fig. 5 A) of Cytokeratin 5 single male.The thymus epithelial precursor cell in these presentation of results human embryo stem cell sources has the certain self in vivo and the ability of differentiation.
Whether the thymus epithelial precursor cell in the human embryo stem cell source of 2, transplanting in the streaming method detection bodies supports the formation of T cell
Antibody information:
The antibody that detects CD4 is the monoclonal antibody in mouse source, and available from biolegend, catalog number is 100407.
The antibody that detects CD8 is the monoclonal antibody in mouse source, and available from biolegend, catalog number is 100705.
With the kidney packing place that is transplanted to nude mice (Beijing Vital River Experimental Animals Technology Co., Ltd.) behind the 14th day the cell harvesting of differentiated system, flow cytometry detects the ratio of nude mice peripheral blood T cells after 1 month.
The flow cytometry step:
1) tail vein blood 3-4 drips, and is dissolved among the 1mlPBS;
2) 4000 change centrifugal 5 minutes;
3) add the 500ul erythrocyte cracked liquid (R&D company, catalog number is: WL2000), on the vortex centrifugal device that cell is all resuspended, room temperature was placed 10 minutes;
4) add the 500ul stop buffer (R&D company, catalog number is: WL2000) centrifugal behind the mixing, 4000 change 5 minutes;
5) the PBS washing is three times, and 4000 change, and centrifugal 5 minutes, abandon supernatant;
6) add the PBS sealing that 200ul contains 0.5%BSA, 4 degree were placed 10 minutes;
7) not centrifugal; Directly with CD4-PE antibody (Biolegend company; Catalog number is 100407) and CD8-FITC (Biolegend company, catalog number is 100705) join in the solution of step 6, the final concentration of antibody is respectively: CD4-PE 2.5ug/ml; CD8-FITC 10ug/ml, 4 degree were placed 30 minutes;
8) the PBS washing is three times, and 4000 change, and centrifugal 5 minutes, abandon supernatant;
9) add the PBS that 200ul contains 2% Paraformaldehyde 96, re-suspended cell, flow type analyzer analysis.
With the nude mice peripheral blood of transplanted cells not is contrast.
The result is shown in Fig. 6 (after transplanting month), and the ratio of nude mice of control group peripheral blood (contrast) the CD4+T cell of transplanted cells is not about 3%, and the ratio of having transplanted the experimental group nude mice peripheral blood CD4+T cell of noble cells is about 7%.The thymus epithelial precursor cell in this presentation of results human embryo stem cell source has certain plastidogenetic ability of support T.
Claims (10)
1. thymus epithelial precursor cell; Be the thymus epithelial precursor cell of the expression epithelial cell adhesion molecule, cytokeratin 5 and the cytokeratin 8 that are obtained by the differentiation of human embryo stem cell or inductive pluripotent stem cell, it has multiplication capacity and to the potential of thymic medulla epithelial cell with differentiation of thymic cortex epithelial cell and/or promotion T cell maturation.
2. thymus epithelial precursor cell according to claim 1 is characterized in that:
Said human embryo stem cell is can be from the human embryonic stem cell of commercial sources acquisition.
3. thymus epithelial precursor cell according to claim 1 and 2 is characterized in that: said can be following any clone from the human embryonic stem cell that commercial sources obtains: BG01, BG02, BG03, BG04, SA01, SA02; SA03, ES01, ES02, ES03, ES04, ES05, ES06; TE03, TE32, TE33, TE04, TE06, TE62, TE07; TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14; The said numbering that is numbered NIH.
4. by the substratum of arbitrary said thymus epithelial precursor cell in human embryo stem cell or the inductive pluripotent stem cell differentiation preparation claim 1 to 3, form by cell culture fluid I, cell culture fluid II and cell culture fluid III,
Said cell culture fluid I prepares according to following method: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, activin A and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol and activin A is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (80-120) ng;
Said cell culture fluid II prepares according to following method: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid, IWP2 and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid and IWP2 is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (0.8-1.2) nmol: (2.3-2.8) nmol;
Said cell culture fluid III obtains according to following method preparation: the basic medium of NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4, wnt3a and cultivation mammalian cell is mixed; Obtain nutrient solution, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4 and wnt3a is 1ml: (0.005-0.05) ml: (0.005-0.05) ml: (0.0005-0.005) ml: (80-120) ng: (80-120) ng.
5. substratum according to claim 4 is characterized in that:
Among the said cell culture fluid I, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol and activin A is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng;
Among the said cell culture fluid II, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, vitamin A acid and IWP2 is 1ml: 0.01ml: 0.01ml: 0.001ml: 1nmol: 2.5nmol;
Among the said cell culture fluid III, the proportioning of the basic medium of said cultivation mammalian cell, NSC 334200 dipeptides, NEAA, 2 mercapto ethanol, bone morphogenetic protein 4 and wnt3a is 1ml: 0.01ml: 0.01ml: 0.001ml: 100ng: 100ng.
6. according to claim 4 or 5 described substratum, it is characterized in that:
The basic medium of said cultivation mammalian cell is X-VIVO 10 substratum.
7. the method with the said thymus epithelial precursor cell of arbitrary said medium preparation among the claim 4-6 comprises the steps:
1) human embryo stem cell or inductive pluripotent stem cell are cultivated on the cell culture fluid I in arbitrary said substratum in claim 4-6;
2) cell of step 1) acquisition is cultivated on the cell culture fluid II in arbitrary said substratum in claim 4-6;
3) cell that step 2) obtains is cultivated on the cell culture fluid III in arbitrary said substratum in claim 4-6, obtains the thymus epithelial precursor cell.
8. preparation method according to claim 7 is characterized in that:
In the step 1), said incubation time is 2.5-3.5 days, and said incubation time was specially 3 days;
Step 2) in, said incubation time is 2.5-4 days, and said incubation time was specially 4 days;
In the step 3), said incubation time is 4.5-5.5 days, and said incubation time was specially 5 days.
9. according to claim 7 or 8 described methods, it is characterized in that:
Said human embryo stem cell is can be from the human embryonic stem cell of commercial sources acquisition.
10. according to arbitrary described method among the claim 7-9, it is characterized in that: said can be following any clone from the human embryonic stem cell that commercial sources obtains: BG01, BG02, BG03, BG04, SA01, SA02; SA03, ES01, ES02, ES03, ES04, ES05, ES06; TE03, TE32, TE33, TE04, TE06, TE62, TE07; TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14; The said numbering that is numbered NIH;
Arbitrary described method or claim 4 or the application of 5 described substratum in preparation thymic medulla epithelial cell and thymic cortex epithelial cell and/or promotion T cell maturation among arbitrary described cell, the claim 6-9 among the claim 1-3.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113646423A (en) * | 2019-04-01 | 2021-11-12 | 纽约市哥伦比亚大学理事会 | Method for promoting differentiation of thymic epithelial cells and thymic epithelial cell progenitors of pluripotent stem cells |
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