CN105002134B - Prepare the composition of pluripotent stem cell, method, kit and application thereof - Google Patents

Abstract

A kind of efficient stable of present invention offer prepares the composition of hyperimmunization defect mammal (such as mouse) pluripotent stem cell, using the method for the composition, the kit and application thereof comprising the composition, and the composition includes：Stem cell basal medium；With vitamin C or derivatives thereof.

Description

Prepare the composition of pluripotent stem cell, method, kit and application thereof

Technical field

A kind of efficient stable of present invention offer prepares hyperimmunization defect mammal (such as mouse) pluripotent stem cell Composition, method, kit and application thereof.

Background technology

In order to improve the understanding to human body hematopoiesis generation, inherent immunity, communicable disease, cancer and regenerative medicine and recognize Know, stem cell field be dedicated to developing all the time humanized mouse model (Bente et al., 2005；King et al., 2008；Kumar et al.,2008；Sun et al.,2007；Yajima et al.,2009).In recent years, in exploit person Many important experiment progress are achieved in terms of the mouse of source, one of them is to establish hyperimmunization defect NOD-scid Il2rg^–/–(NPG) mouse model, this mouse model have severe immune deficiency (the severe combined Immunodeficiency, SCID) and features (the Ito et such as interleukin-2Rg (Il-2Rg) deletion allele al.,2002).The genetic background for the non-obese patients with type Ⅰ DM immune deficiency that NPG mouse have leads to dendritic cell function and huge Thermophilic cell function is impaired so that defect (Shultz et al., 1995) occurs for inherent immunity, while Prkdc gene codes produce Gene also has occurred on NPG mouse in the raw key protein as regulation and control V (D) J recombinations in T cell and B cell maturation Mutation leads to eliminate acquired immunity function (Blunt et al., 1995).In addition, Il2rg^–/–Rite-directed mutagenesis is deleted completely Participate in IL-2R gamma chains (the 2 receptor gamma of interleukin of regulation and control immune function process Chain) so that participate in Mice Body the process of regulation and control heteroplastic transplantation human cell NK cells extremely missing (Cao et al., 1995).Based on this, these immune deficiency advantages make NPG mouse as the pole of human cell, tissue, organ is studied in vivo Good receptor model, the especially people of the offer important clue being established as research Human biology and applied to preclinical study Be even more in terms of source mouse model played important function (Devoy et al., 2012；Koo et al.,2009；Sato et al.,2011)。

Although NPG mouse are hyperimmunization defects, and the mouse humanized human diseases of research in vivo of NPG is proved to be Very valuable animal model, but still have the shortcomings that some apparent hinder its future and be widely used in establishing people source Change mouse model.For example, lacking the suitable MHC molecule and some raising human bodies of screening T cell in the thymus gland of NPG mouse The special growth factor of the people of cell survival and growth.Resist in addition, NPG mouse are presented low-level and changeable T cell and rely on Precursor reactant (Shultz et al., 2007).The above disadvantage may hinder it to be further applicable to establish humanization mouse The fields such as model.

However, a nearest research group reports the functionality for obtaining human embryo stem cell source in NPG mouse Thymus gland organoid, after being transplanted for research the maturation of human T-cell provide valuable clue and thinking (Sun et al., 2013).In addition, the strategy of another exploitation more suitably humanization mouse is to import encoding human into the genome of NPG mouse The gene (Shultz et al., 2007) of the growth factor of T cell screening and survival after the special HLA of class and regulation and control transplanting.So And this implementation of strategies depends on and establishes pluripotent stem cell from NPG mouse in vitro.Therefore external to establish stable NPG Mouse pluripotent stem cell system is exactly an extraordinary research point of penetration.

Invention content

The present invention provides the sides that a kind of external efficient stable establishes hyperimmunization defect NPG mouse pluripotent stem cells Method, this method establish one kind and adding Vitamin C on the basis of traditional 2i/LIF condition of culture by small molecule Screening Platform Condition of culture, to make the hyperimmunization defect NPG mouse for being unable to stabilization in vitro culture under traditional 2i/LIF condition of culture Pluripotent stem cell obtains stablizing long-term cultivation.According to the present invention, NPG Mouse Blastocysts are detached first, and are inoculated in small Mouse is cultivated 5-8 days on fiber feeder cells under traditional 2i/LIF condition of culture or under the conditions of 2i/LIF+Vitamin C, Then it is digested to unicellular passage with pancreatin or accutase.After passage using 2i/LIF+Vitamin C carry out long-term cultivation or Passage.This method success establishment efficiency may be up to 50% and (obtain 14 plants of pluripotent stem cells from 28 pieces of NPG Mouse Blastocysts System).

The hyperimmunization deficient mice pluripotent stem cell system that this method is established has the institute of mouse pluripotent stem cell There is characteristic feature and carries the characteristic gene mutation of NPG mouse.Therefore, the present invention establishes a kind of external efficient stable and builds The method of vertical hyperimmunization deficient mice pluripotent stem cell carries for the completely new more good humanized mouse model of the following exploitation The operating platform stablized is supplied.

More specifically, the present invention provides the following terms：

1. a kind of composition for the pluripotent stem cell being used to prepare mammal, the composition include：Stem cell base Basal culture medium；With vitamin C or derivatives thereof, wherein

(1) the preferably mammal is immune deficiency mammal, more preferably immunodeficient mouse, is most preferably selected The immunodeficient mouse of the group formed from following Strains of Mouse：NOD-scid Il2rg^–/–(NPG), NOD-scid, B2m^-/-, NOD-scidB2m^-/-,Tg(HLA-A2),NOD-scidTg(HLA-A2),BALB/c-Rag2^-/-Il2rg^-/-,Rag1^-/-,NOD- scid IL2rg^-/-,IL2rg^-/-,NOD-Rag1^-/-Il2rg^-/-, NOD-Rag1^-/-,Prf1^-/-,NOD-Rag1^-/-Prf1^-/-,Tg (SOD1-G93A)1Gur,Dmd^mdx-5Cv,NOD-Rag1^-/-Tg(SOD1-G93A)1Gur,NOD-Rag1^-/-Ins2^Akita, Ins2^Akita,NOD-Rag1^-/-Dmd^mdx-5Cv；

(2) the preferably stem cell basal medium is the culture medium used under traditional 2i/LIF condition of culture, more preferably Including the stem cell media selected from group consisting of：D/F12, K-DMEM, KSR, FBS, N2, B27 and combination thereof； And/or

(3) preferably the composition includes selected from the vitamin C or derivatives thereof by following presentation：

2. according to the composition described in above 1, wherein the composition includes other signal path regulatory factor or connection Other signal path regulatory factor is closed to use, the preferably described signal path regulatory factor be selected from WNT signal paths regulatory factor, LIF/STAT3 accesses regulatory factor, MAPK accesses inhibiting factor, vitamin C associated downstream signal path regulatory factor and they Combination, preferably GSK3-beta inhibitor or MAPK inhibitor, the preferably described regulatory factor act on above-mentioned signal path Cell factor, chemical small molecule, transcription factor or extracellular matrix.

3. according to the composition described in above 2, wherein the composition includes：knock-out DMEM+N2(100x)/ B27 (50x), dual anti-(penicillin/streptomycin), beta -mercaptoethanol, glutamine, nonessential amino acid (Non-essential Amino Acids), CHIR99021, PD0325901, LIF and vitamin C.

4. a kind of kit for the pluripotent stem cell being used to prepare mammal, the kit includes according to the above 1- Composition described in any one of 3 includes also optionally other stem cell inducible factor, and the preferably described mammal is immune Defect mammal, more preferably immunodeficient mouse.

5. a kind of method for the pluripotent stem cell being used to prepare mammal, the method includes according to the above 1-3 Any one of described in composition in the presence of cultivate initial mammalian cell, the preferably described initial mammalian cell be feed The embryonic stem cell or be suitable for obtaining pluripotent stem cell by reprogramming technology that newborn animal body early embryo (blastaea) is established Body cell uses the method wherein for body cell in the later stage for obtaining pluripotent stem cell by reprogramming technology, It is preferred that the mammal is immune deficiency mammal, more preferably immunodeficient mouse；It is preferred that the culture is that having feeding It supports coating systems or is cultivated under coating systems without raising.

6. the mammal pluripotent stem cell obtained by the method according to above 5, the preferably described mammal It is immune deficiency mammal, more preferably immunodeficient mouse, most preferably NPG mouse, the preferably described pluripotent stem cell tool One or more of standby following property is whole：

(1) multipotency correlating markings gene, such as endogenous Oct4, Sox2, Nanog, Sall4, Dnmt3b, Dppa2 are expressed, Cripto etc.；

(2) alkaline phosphatase staining strong positive, expression various kinds of cell in and cell membrane surface multipotency correlating markings egg In vain, such as intracellular OCT4, SOX2, NANOG, the SSEA-1 etc. of cell membrane surface；

(3) there is the ability for forming teratoma, teratoma can detect triploblastica cell；

(4) Long Term Passages (>15 generations) after still have normal karyotype；

(5) has chimeric or germline transmission capacity；

(6) mutation of hyperimmunization dcc gene is carried, as PRKDC and IL2RG functional deficiencies are mutated；With

(7) pluripotent stem cell source can be the embryonic stem cell established from body early embryo (blastaea), or pass through The inducible pluripotent stem cell that reprogramming method obtains.

7. a kind of method producing mammalian animal model, the preferably described mammal is immune deficiency mammal, more excellent Choosing is immunodeficient mouse, the method includes：The mammal is injected with the pluripotent stem cell according to above 6 In embryo's (such as 8 cell stages) or blastaea；And/or the embryo of acquisition or blastaea are transplanted to and continue to develop in the mammalian body At chimeric mammal.

8. according to the method described in above 7, wherein the mammal is inhuman primate mammal.

9. according to the method described in above 7 or 8, wherein the pluripotent stem cell is by further genetic modification.

10. the mammalian animal model that the method according to any one of above 7-9 obtains, preferably immune deficiency lactation are dynamic Object model, more preferably immunodeficient mouse model, most preferably immune deficiency NPG mouse models.

Description of the drawings

Fig. 1 is NPG murine genes type used identification and immune deficiency Functional Characterization.Figure 1A reflects for NPG murine genes types It is fixed.The result shows that compared with wild-type mice (WT), NPG mouse carry the mutation of PRKDC and IL2RG functional deficiencies.Figure 1B For NPG mouse immune defect Functional Characterizations.The result shows that compared with control group mice, T, B and work(are lacked in NPG Mice Bodies It can property NK cells.

Fig. 2 is the NPG mouse pluripotent stem cells system form obtained, Preliminary Identification and Efficiency Statistics.Fig. 2A (left side) be from The protrusion that NPG Mouse Blastocysts are grown, (in) to grow two days colony morphologies after unicellular passage, (right side) is AP colored graphs； Fig. 2 B are NPG Establishing Mouse Embryonic Stem Cell Line Efficiency Statistics in 2 batches of experiments.Scale:100μm.

Fig. 3 is the growth characteristics of NPG mouse pluripotent stem cells system and the Genome stability identification after Long Term Passages.Figure 3A is different pluripotent stem cell systems, including 2 plants of NPG embryonic stem cell lines, 1 plant of wild type mouse embryos stem cell line and 1 plant The growth curve chart of the wild type pluripotent stem cell system of chemical small molecule induction；Fig. 3 B are the NPG Development of Mouse Embryos for being passaged to for 15 generations The caryogram of tire stem cell line is identified.Illustrate that NPG mice embryonic stem cell systems remain to keep genome steady in Long Term Passages It is qualitative.

Fig. 4 identifies for NPG mouse pluripotent stem cell body outer properties.Fig. 4 A reflect for NPG mice embryonic stem cell system groupizations It is fixed, show that Oct4, Sox2, Nanog, SSEA-1 are the positive；Fig. 4 B are RT-PCR as a result, showing NPG mice embryonic stem cell systems Express dryness gene；Fig. 4 C are DNA methylation assay as a result, showing the regions Nanog promoter of NPG mice embryonic stem cell systems Height demethylation.Scale:100μm.

Fig. 5 is Property Identification in NPG mouse pluripotent stem cell bodies.Fig. 5 A teratomas are the result shows that NPG mouse embryonic stems Cell line can break up to ectoderm, mesoderm and entoderm；Fig. 5 B illustrate that NPG mouse embryo stem cells have and generate gomphosis mouse Ability.Scale：100μm.

Fig. 6 is NPG mouse pluripotent stem cell expression pattern analysis.Fig. 6 A and 6B clusterings show NPG mouse embryonic stems Cellular gene expression level is similar to wild type embryos stem cell line and pluripotent stem cell system, with mouse embryonic fibroblasts There were significant differences；Fig. 6 C are that GO analyzes (david.abcc.ncifcrf.gov/), are shown in NPG mouse embryo stem cells The signal path that reconciliation is lowered.

Fig. 7 is that NPG mouse pluripotent stem cells system carries the characteristic detection in Gene Mutation of NPG mouse.The result shows that same Wild type embryos stem cell line is compared with pluripotent stem cell system, and NPG mouse pluripotent stem cells carry PRKDC and IL2RG Functional deficiency is mutated.IL2RG:Mouse IL2RG genomes detect；PRKDC:Mouse PRKDC genomes detect；GAPDH:Internal reference Gene.

Fig. 8 is the primer list

Specific implementation mode：

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The main agents and instrument used in experiment

(1) pluripotent stem cell system is established from NPG Mouse Blastocysts

Hyperimmunization deficient mice source and ore grade indexes：

The hyperimmunization deficient mice strain that the present embodiment is selected is provided by Beijing Weitongda Biotechnology Co., Ltd. NPG mouse.There is similar gene mutation and immune deficiency with internationally recognized hyperimmunization deficient mice strain NSG mouse Feature.Testing result is shown in attached drawing 1.

Main reagent to be used：

NPG mouse pluripotent stem cell culture mediums：Knock-out DMEM+N2 (100x)/B27 (50x), 1% is dual anti-(green Mycin/streptomysin), 0.1mM beta -mercaptoethanols, 1mM glutamine, 1% nonessential amino acid (Non-essential Amino Acids), 3 μM of CHIR99021,0.5 μM of PD0325901, Vitamin C (50 μ g/ml) LIF (10ng/ml).

Experimental procedure：

1) the NPG Mouse Blastocysts (being provided by Beijing Weitongda Biotechnology Co., Ltd.) in embryo's E3.5 periods are provided.

2) NPG Mouse Blastocysts are inoculated on mouse fibroblast feeder layer cell, in NPG pluripotent stem cell culture mediums Culture 5-8 days in (2i/LIF+Vitamin C), replace fresh culture daily therebetween.

3) clone that Primary Growth comes out is chosen by hand and becomes unicellular with pancreatin or accutase digestion, passage connects Kind on fresh mouse fibroblast feeder layer cell, in NPG mouse pluripotent stem cell culture mediums (2i/LIF+Vitamin C culture and later passages are carried out in).It is 4-6 days to pass on the period.

4) cell line that select to pass on steadily in the long term carries out follow-up identification experiment.

(2) culture of NPG mouse pluripotent stem cell system and passage

Main reagent to be used：

NPG mouse pluripotent stem cell culture mediums：Knock-out DMEM+N2 (100x)/B27 (50x), 1% is dual anti-(green Mycin/streptomysin), 0.1mM beta -mercaptoethanols, 1mM glutamine, 1% nonessential amino acid (Non-essential Amino Acids), 3 μM of CHIR99021,0.5 μM of PD0325901, Vitamin C (50 μ g/ml), LIF (10ng/ml).

Experimental procedure：

1) observation NPG mouse pluripotent stem cells clone waits for that its length to logarithmic phase (3-5 days) can be passed on.

2) culture medium in original 10cm culture dishes is sucked out, is washed 2 times with PBS, to remove upper layer dead cell.1-2ml is added Accutase digests 1-3 minutes in 37 degrees Celsius.It constantly gently pats therebetween and shakes culture dish.

3) wait for that NPG mouse pluripotent stem cells clone starts to scatter and has about 50% or more clone from feeder cells When falling off, about 2-3ml NPG mouse pluripotent stem cell culture mediums are added and terminate digestion.

4) gently the clone not fallen off is blown off with liquid-transfering gun from feeder cells, is transferred to 15ml centrifuge tubes together In.

5) it is gently blown and beaten with liquid-transfering gun, clone is made to become unicellular.900rpm abandons supernatant after centrifuging 3 minutes, about 2- is added 3ml NPG mouse pluripotent stem cell culture mediums clean, and 900rpm is centrifuged 3 minutes.

6) ready feeder cells will be shifted to an earlier date to be washed one time with PBS, it is NPG mouse pluripotent stem cell cultures to change liquid Base.

7) NPG mouse pluripotent stem cells clone's suspension is connected on feeder cells and is shaken up by suitable density, be placed in It is cultivated in 37 degrees Celsius of incubators.Culture medium is replaced to passage next time (after 3-5 days) daily later or is frozen.

Experimental result is shown in Fig. 2-Fig. 3, Fig. 7, the NPG mouse pluripotencies established with NPG mouse pluripotent stem cell culture mediums Stem cell can maintain steadily in the long term in vitro.

(3) NPG mouse pluripotent stem cell general survey method

1. alkaline phosphatase activities dyeing identification

Main reagent to be used：

Alkaline Phosphatase Detection Kit

PBS buffer solution

Experimental procedure：

1) it after removing cell culture medium, is washed 3 times with PBS.

2) Kit operation instructions are pressed to operate.(http://www.millipore.com/catalogue/item/SCR004#)

2. cellular immunofluorescence is identified

Main reagent to be used：

PBS buffer solution

4% paraformaldehyde

Confining liquid：0.2%Triton X100 and final concentration of 5% 2 antiserum are added in PBS buffer solution

Antibody diluent：The BSA of PBS buffer solution+0.1%

DAPI solution：1μg/ml

Experimental procedure：

1) it after removing cell culture medium, is washed 3 times with PBS.

2) it is fixed with 4% paraformaldehyde.(room temperature 20 minutes or 4 C overnights)

3) paraformaldehyde is sucked out, is washed 3 times with PBS buffer solution, it is every all over 5 minutes.

4) confining liquid closing is added.(room temperature 60 minutes or 4 C overnights)

5) confining liquid is sucked out, is washed 3 times with PBS buffer solution, it is every all over 5 minutes.

6) primary antibody (Oct4 (Abcam), Nanog (R＆D), Sox2 (Santa Cruz), SSEA-1 (millipore)) is added (press 1：50-1：200 are diluted).(37 degrees Celsius 2 hours or 4 C overnights)

7) primary antibody is sucked out, is washed 3 times with antibody diluent, it is every all over 5 minutes.

8) secondary antibody (donkey anti rabbit rhodamine (Santa Cruz), donkey anti goat is added rhodamine(Santa Cruz),goat anti mouse rhodamine(Santa Cruz).(press 1：50-1：200 carry out Dilution).(in being protected from light 37 degrees Celsius of place 2 hours or 4 C overnights)

9) secondary antibody is sucked out, is washed 3 times with antibody diluent, it is every all over 5 minutes.

10) DAPI solution is added and contaminates nucleus, contaminated 1-5 minutes in room temperature.

11) DAPI solution is sucked out, is washed 3 times with antibody diluent, it is every all over 5 minutes.

12) be added antibody diluent, fluorescence microscopy under the microscope or be kept in dark place in 4 degrees Celsius.

3. cell total rna extracts

Main reagent to be used：

Trizol reagents

Isopropanol

Chloroform

DEPC water

75% ethyl alcohol：DEPC water is prepared

Absolute ethyl alcohol

Experimental procedure：

1) it after removing cell culture medium, is washed 3 times with PBS.

2) addition and the isometric Trizol reagents of culture medium, are placed at room temperature for 10 minutes.Cell pyrolysis liquid is transferred to In 1.5ml centrifuge tubes.

3) chloroform (volume is the 1/4 of Trizol) acutely concussion about 15 seconds are added.It is placed at room temperature for 2-3 minutes.

4) it is centrifuged 10 minutes in 4 degrees Celsius, centrifugal force 12000g.

5) it shifts in supernatant to new 1.5ml centrifuge tubes, isometric isopropanol is added, is placed at room temperature for 10 minutes.

6) it is centrifuged 10 minutes in 4 degrees Celsius, centrifugal force 12000g.

7) it discards supernatant, 75% ethyl alcohol 0.5ml is added and washes 1-2 times

8) it is centrifuged 5 minutes in 4 degrees Celsius, centrifugal force 7500g.

9) it discards supernatant, centrifuge tube is inverted on blotting paper and dries ethyl alcohol.

10) 10 μ l of DEPC water are added in 37 degrees Celsius of dissolvings.

4.RNA reverse transcriptions are cDNA

Main reagent to be used：

CDNA reverse transcription reagent box A3500 (Promega)

Experimental procedure：

1) configuration reaction system (20 μ l)

2) this system mixed liquor is placed at room temperature for 10 minutes, is then placed in PCR instrument, 42 degrees Celsius are incubated 15 minutes, and 95 Degrees Celsius 5 minutes.

3) obtained cDNA solution measured concentrations are placed on -20 degrees Celsius of preservations after reaction terminating.

5. cell genomic dna extracts

Main reagent to be used：

Cell pyrolysis liquid：100ml systems 100 μ g/ml, 0.2%SDS, 20mM EDTA, 10mM Tris containing Proteinase K, 0.1M NaCl

Experimental procedure：

1) it is resuspended in after digesting cell with pancreatin in precooling PBS.

2) cell is collected within 10 minutes in 4 degrees Celsius of centrifugations.Centrifugal force is 1500rpm

3) 400 μ l of cell pyrolysis liquid are added, 50 degrees Celsius shake 1 hour

4) after system is cooled to room temperature, 1/10 volume is added and is saturated NaCl, overturn mixing repeatedly about 10 minutes.

5) centrifuge tube is placed in cooled on ice about 20-30 minutes.

6) it is centrifuged 15 minutes in 4 degrees Celsius.Centrifugal force is 4000g

7) supernatant is transferred in new 1.5ml centrifuge tubes, the absolute ethyl alcohol being pre-chilled in equal volume, mixing is added.

8) centrifuge tube is placed in cooled on ice about 20 minutes.

9) it is centrifuged 15 minutes in 4 degrees Celsius.Centrifugal force is 13000rpm

10) supernatant is sucked out, is precipitated in room temperature aeration-drying genomic DNA.

11) 30-50ul sterile waters are added to carry out staying overnight dissolving.

6. polymerase chain reaction (PCR) is analyzed

Main reagent to be used：

2 × EasyTaq PCR Supermix (Beijing Quanshijin Biotechnology Co., Ltd)

Experimental procedure：

1) configuration reaction system (200 μ l)

2) reaction condition is set

3) PCR reaction products are tested with agarose gel electrophoresis

7. real-time quantitative polymerase chain reaction (RT-PCR analyses)

Main reagent to be used：

Cell pyrolysis liquid：100ml systems 100 μ g/ml, 0.2%SDS, 20mM EDTA, 10mM Tris containing Proteinase K, 0.1M NaCl

Experimental procedure：

PCR uses Power on 7300 Sequence Detection System of ABI PrismGreen PCR Master Mix (Applied Biosystems) system completes data analyses and uses delta-delta Ct methods.

8. teratoma is tested

Experimental procedure：

1) target cell for being intended to carry out teratoma injection is resuspended in D/F12 culture mediums, by the cell of one piece of 6cm culture dish A corresponding site injection enters NOD/SCID immunodeficient mouses (Beijing Weitongda Biotechnology Co., Ltd.) abdominal cavity or subcutaneous.

2) occur putting to death the mouse neck that breaks after apparent teratoma generates after transplantation site.It is taken with dissection key and dissecting forceps separation Go out teratoma.

3) teratoma is placed in 4% paraformaldehyde and is fixed, specimens paraffin embedding slices dyeing observation.

9.NPG mouse pluripotent stem cells inject 8 cell stage of C57 mouse or the chimeric experiment of blastula embryo

Experimental procedure：

7-10 NPG mouse pluripotent stem cell microinjection (ties up the sensible limited public affairs of biotechnology in Beijing to C57 mouse Department) (isolated in 2.5 days (2.5dpc) female mices of post-coitum) or NPG mouse pluripotent stem cells are micro- in 8 cell stages It is injected into C57 Mouse Blastocysts (isolated in 3.5dpc female mices), after cultivating one day, blastaea or 8 cell stages are transplanted respectively Into 2.5dpc or 0.5dpc CD-1 false pregnancy female mice bodies, (6-8 embryo in each fallopian tubal or cornua uteri) enables it continue to develop Birth is gomphosis mouse.

10.NPG mouse pluripotent stem cell NPG characteristic gene saltant types detect

Experimental procedure：

1) preparation of genomic DNA

See：In " (one) NPG mouse pluripotent stem cell general surveys method " " 5. extracting genome DNA "

2) PRKDC point mutation PCR reaction systems (10 μ l)

3) IL2RG knocks out PCR reaction systems (10 μ l)

4) reaction condition is set

11.NPG mouse pluripotent stem cell karyotypings

It is karyotype analytical procedure (Beijing Weitongda Biotechnology Co., Ltd.'s product description) that the present invention, which uses,.

12.RNA-SEQ is analyzed

It is conventional RNA-SEQ analytical procedures that the present invention, which uses, builds library and uses Illumina mRNA-seq Prep Kit (Illumina).Sequencing uses Illumina HiSeq 2500 (Illumina).

Experimental result is shown in Fig. 4-Fig. 7, the NPG mouse pluripotencies established with NPG mouse pluripotent stem cell culture mediums are dry thin Born of the same parents have all characteristic features of mouse pluripotent stem cell.

Therefore, the present invention includes following aspect：

1. obtained NPG mouse pluripotent stem cells have following property：

(1) multipotency correlating markings gene, such as endogenous Oct4, Sox2, Nanog, Sall4, Dnmt3b, Dppa2 are expressed, Cripto etc..

(2) alkaline phosphatase staining strong positive, expression various kinds of cell in and cell membrane surface multipotency correlating markings egg In vain, such as intracellular OCT4, SOX2, NANOG, the SSEA-1 etc. of cell membrane surface.

(3) there is the ability for forming teratoma, teratoma can detect triploblastica cell.

(4) Long Term Passages (>15 generations) after still have normal karyotype.

(5) has chimeric or germline transmission capacity.

(6) mutation of hyperimmunization dcc gene is carried, as PRKDC and IL2RG functional deficiencies are mutated.

(7) NPG mouse pluripotent stem cell source can be the embryonic stem cell established from body early embryo, or pass through The inducible pluripotent stem cell that reprogramming method obtains.

Wherein meet at least one in (5) (6) (7) item.

2. the method for obtaining hyperimmunization deficient mice pluripotent stem cell：

(1) initial cell source：It can be the embryonic stem cell established from hyperimmunization deficient mice body early embryo, also may be used It is the hyperimmunization deficient mice body cell suitable for reprogramming technology, by the reprogramming of foreign gene mediation, or by chemistry The method that small molecule etc. does not depend on foreign gene is reprogrammed, and is used condition of the present invention in the later stage, is obtained property described in 1 Cell.Hyperimmunization deficient mice cell origin is not limited only to NPG Strains of Mouse, further includes having similar hyperimmunization defective The NOD-scid of matter, B2m^-/-,NOD-scidB2m^-/-,Tg(HLA-A2),NOD-scidTg(HLA-A2),BALB/c-Rag2^-/- Il2rg^-/-,Rag1^-/-,NOD-scid IL2rg^-/-,IL2rg^-/-,NOD-Rag1^-/-Il2rg^-/-, NOD-Rag1^-/-,Prf1^-/-, NOD-Rag1^-/-Prf1^-/-,Tg(SOD1-G93A)1Gur,Dmd^mdx-5Cv,NOD-Rag1^-/-Tg(SOD1-G93A)1Gur,NOD- Rag1^-/-Ins2^Akita,Ins2^Akita,NOD-Rag1^-/-Dmd^mdx-5CvStrains of Mouse etc..

(2) used signal path regulatory factor group during obtaining hyperimmunization deficient mice pluripotent stem cell It closes.Relate generally to the combination between following signals access：It is crucial that WNT signal path regulatory factors, LIF/STAT3 accesses Regulatory factor, MAPK accesses inhibiting factor and Vitamin C associated downstream signal path regulatory factors.Regulatory factor type can To be cell factor, chemical small molecule, transcription factor, extracellular matrix etc. can act on the substance of above-mentioned signal path.

For example, the present invention can use following GSK3-beta inhibitor：

For example, the present invention can use following MAPK inhibitor：

For example, the present invention can use following Vitamin C and its derivative：

(3) basic culture solution includes D/F12, K-DMEM, KSR, and FBS, N2, B27 etc. is suitable for convenient stem cells culture Combination between culture solution.Such as wherein typical concentrations proportioning is D/F12：KSR=9：1–3：1, K-DMEM：KSR=9：1–3： 1, D/F12：FBS=9：1–3：1, K-DMEM：FBS=9：1–3：1.The conventional amount used addition that N2, B27 are suggested by manufacturer.

(4) according to a kind of above-mentioned hyperimmunization deficient mice pluripotent stem cell culture medium for designing use above： Knock-out DMEM+N2 (100x)/B27 (50x), 1% dual anti-(blueness/streptomysin), 0.1mM beta -mercaptoethanols, 1mM paddy ammonia Amide, 1% nonessential amino acid (Non-essential Amino Acids), 3 μM of CHIR99021,0.5 μM PD0325901, Vitamin C (50 μ g/ml), LIF (10ng/ml).

Wherein basic culture solution condition is replaced by described in (3) and arbitrarily combines.

(5) above-mentioned hyperimmunization deficient mice pluripotent stem cell medium culture base use scope：Hyperimmunization defect Mouse pluripotent stem cell induces, including builds from body early embryo and to be and the phase process after reprogramming of somatic cells.Processing time Generally 5-7 days.Hyperimmunization deficient mice pluripotent stem cell routine culture includes raising coating systems and is trained without feeder layer The system of supporting.

3. the application of hyperimmunization deficient mice pluripotent stem cell

The hyperimmunization deficient mice pluripotent stem cell with property described in 1 obtained using the method described in 2 It can be applied to following aspect：

(1) hyperimmunization deficient mice animal model is produced.

(2) genetic modification is carried out to this hyperimmunization deficient mice pluripotent stem cell, including but not limited to uses and turns Gene, TALEN, CRISPER/CAS9, the methods of ZFN.

(3) new immune deficiency is generated by carrying out genetic modification to this hyperimmunization deficient mice pluripotent stem cell Mouse model.

(4) the hyperimmunization deficient mice animal model produced using the above method can be applied to human archeocyte or tissue Transplanting, oncobiology, humanized antibody manufacture, human infectious disease's research, hematopoiesis and immunological investigation, new humanized animal The research fields such as Model R ＆ D.

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Claims (12)

1. stem cell basal medium and vitamin C or derivatives thereof a kind of being used to prepare immune deficiency mammal preparing Application in the composition of pluripotent stem cell, wherein

(1) the immune deficiency mammal is NOD-scid Il2rg^–/–(NPG) immunodeficient mouse；

(2) the stem cell basal medium is the culture medium used under traditional 2i/LIF condition of culture；With

(3) composition includes selected from the vitamin C or derivatives thereof by following presentation：

2. application according to claim 1, wherein the stem cell basal medium is comprising selected from group consisting of Stem cell media：D/F12, K-DMEM, KSR, FBS, N2, B27 and combination thereof.

3. application according to claim 1, wherein the composition includes other signal path regulatory factor or joint Other signal path regulatory factor uses.

4. application according to claim 3, wherein the signal path regulatory factor be selected from WNT signal paths regulate and control because Son, LIF/STAT3 accesses regulatory factor, MAPK accesses inhibiting factor, vitamin C associated downstream signal path regulatory factor and it Combination.

5. application according to claim 3, wherein the signal path regulatory factor is GSK3-beta inhibitor or MAPK Inhibitor.

6. application according to claim 3, wherein the signal path regulatory factor is the cell for acting on signal path The factor, chemical small molecule, transcription factor or extracellular matrix.

7. application according to claim 3, wherein the composition includes：Knock-out DMEM+N2/B27, penicillin And streptomysin, beta -mercaptoethanol, glutamine, nonessential amino acid, CHIR99021, PD0325901, LIF and vitamin C.

8. the composition described in claim any one of 1-7 is preparing a kind of pluripotent stem cell being used to prepare mammal Kit in application, the kit includes also optionally other stem cell inducible factor, and the mammal is NOD-scid Il2rg^–/–(NPG) immunodeficient mouse.

9. a kind of method for the pluripotent stem cell being used to prepare immune deficiency mammal, the method includes in claim Initial mammalian cell is cultivated in the presence of composition described in any one of 1-7, the initial mammalian cell is to feed The embryonic stem cell or be suitable for obtaining the body of pluripotent stem cell by reprogramming technology that newborn animal body early embryo blastaea is established Cell uses the method, institute wherein for body cell in the later stage for obtaining pluripotent stem cell by reprogramming technology It is NOD-scid Il2rg to state immune deficiency mammal^–/–(NPG) immunodeficient mouse；The culture is that having feeder layer body System cultivates without raising under coating systems.

10. according to the method described in claim 9, the wherein described pluripotent stem cell has one or more of following property Or all：

(1) multipotency correlating markings gene is expressed, the multipotency correlating markings gene is selected from endogenous Oct4, Sox2, Nanog, Sall4, Dnmt3b, Dppa2 and Cripto；

(2) alkaline phosphatase staining strong positive, expression various kinds of cell in and cell membrane surface multipotency correlating markings albumen, institute It states marker protein and is selected from intracellular OCT4, the SSEA-1 of SOX2, NANOG and cell membrane surface；

(3) there is the ability for forming teratoma, teratoma to be able to detect that triploblastica cell；

(4) Long Term Passages, i.e.,>Still has normal karyotype after 15 generations；

(5) has chimeric or germline transmission capacity；

(6) mutation of hyperimmunization dcc gene is carried, it is functional that the immune deficiency gene mutation is selected from PRKDC and IL2RG Deletion mutation；

(7) pluripotent stem cell source is the embryonic stem cell established from body early embryo blastaea, or to pass through reprogramming method The inducible pluripotent stem cell obtained.

11. a kind of method producing immune deficiency mammalian animal model, the immune deficiency mammal is NOD-scid Il2rg^–/–(NPG) immunodeficient mouse, the method includes：It is immune with being obtained by the method according to claim 11 The pluripotent stem cell of defect mammal injects in embryo or the blastaea of the immune deficiency mammal；And/or it will obtain Embryo or blastaea be transplanted to and continue to develop into chimeric mammal in the mammalian body.

12. according to the method for claim 11, wherein the pluripotent stem cell is by further genetic modification.