CN104946581A - Special culture medium and method for culturing porcine trophoderm stem cells - Google Patents
Special culture medium and method for culturing porcine trophoderm stem cells Download PDFInfo
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Abstract
The invention discloses a special culture medium and method for culturing porcine trophoderm stem cells. The special culture medium is composed of bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol. The culture method comprises the following steps: previously coating a stem cell culture dish with an extracellular matrix; digesting porcine blastula with proteinase to remove the zona pellucida, transferring into the stem cell culture dish, and adding the special culture medium; and culturing until a trophoderm stem cell cloning group with the total cell count of 1000-2000 is formed, digesting into unicells, transferring into a new extracellular-matrix-coated culture dish, and carrying out cell subculture. The special culture medium has the advantages of definite components and high use safety, and avoids the pollution of heterogenous cells. The culture method is simple and efficient, and has the advantages of high cloning formation rate, low apoptosis rate, high proliferation and subculture capacity, high safety and high stability when being used for porcine trophoderm stem cell culture.
Description
Technical field
The invention belongs to stem cells technology research field, be specifically related to a kind of special culture media and method of cultivating pig cytotrophoblast stem cells.
Background technology
Stem cell is the original undifferentiated cell that a class has multi-lineage potential and the of self-replication capacity, is the initiating cell of each histoorgan forming mammal.Medical circle is called " general-purpose cell ".
There is first time cytodifferentiation to during blastaea in animal embryo early development, forms the different inner cell mass of two kinds of Characteristic and function and trophoderm.Cytotrophoblast stem cells is the progenitor cell of placenta tissue cell, is progressively divided into trophoderm precursor cell, chorionic trophoblast, syntrophoblast and polykaryocyte etc.In the epigenetic reprogramming process that fetal development is early stage, the relevant enzyme acting in conjunction such as DNMTs, TETs, HDACs maintains the chromatin condensation of the overall hypomethylation level of trophocyte's genome and chorionic trophoblast, participate in regulating trophoblastic differentiation, migration and the endometrial ability of intrusion (Logan et al., 2013).In order to disclose molecular mechanism so complicated in trophoderm differentiation and development, from body early embryo separation and purification cytotrophoblast stem cells, the model setting up its vitro culture is necessary.Successfully be separated from Mouse Blastocysts trophectoderm the earliest and obtain cytotrophoblast stem cells; they can maintain self under the environment adding Prostatropin bFGF and human leukemia inhibitory factor hLIF; each position into mouse placenta can be fitted together to after being injected into Mouse Blastocysts; and be not present in (Tanaka et al., 1998 in each germinal layer of mouse fetal; Takahashi and Yamanaka., 2006; Rielland et al., 2008).The trophocyte system of some other animal also sets up, and comprise ox, sheep and pig etc., these cells can carry out continuous passage cultivation, show epithelial feature.But, there is many unknown difference with the relevant key signal path of renewal and mouse in the cell proliferation of the cytotrophoblast stem cells of ungulates large animal, the culture system of the Mouse trophoblast stem cell of these classics is not also suitable for the separation and Culture of the cytotrophoblast stem cells of the large animal such as domestic animal and primates.There is larger difference in the somatomedin needed for stem cell separation and Culture of various animals and regulatory pathway, it is the report identified with stem cell properties that the cytotrophoblast stem cells that the early blastocyst that there is no pig at present is both at home and abroad originated successfully is built.The primate such as people and rhesus monkey obtains the factor of difficulty due to body early embryo, and the cytotrophoblast stem cells culture system research of groping to be suitable for will spend the longer time.This is to still successfully to build without domestic animal truly and the embryonic stem cell of primates the situation being so far similar.
Acquisition can be separated in the embryo of cytotrophoblast stem cells after mouse prenidation phase with implantation, but, there is no both at home and abroad in ungulates such as being reported in pig, ox and sheep and also can obtain this cytotrophoblast stem cells can bred renewal, there is versatility be similar in mouse.The trophocyte system in pig placenta source sets up, and these cells can carry out continuous passage cultivation, show epithelial feature, express differentiation marker Lamin A/C, but do not have multipotential stem cell feature (Flechon et al., 1995; La Bonnardiere et al., 2002).Song Zhi waits by force (2013) to use mouse fetal inoblast as feeder layer cells, in basic culture solution, add fibroblast growth factor FGF4 and human leukemia inhibitory factor LIF, from the female blastaea of orphan of the 6th day, be separated to pig cytotrophoblast stem cells like cell.In its test, culture efficiency is low, is about 30 blastaeas and can obtains 1 cell clone group, morphologically embody cytotrophoblast stem cells feature, but fail to prove its stem cell properties, that is also there is no real pig cytotrophoblast stem cells.Li Rongfeng etc. (2013) have invented a kind of ox cytotrophoblast stem cells and have built system, method, be ox blastaea is inoculated in the feeder layer of mouse fetal inoblast and Wnt-3A mouse subcutaneous connective tissue cell, in nutrient solution, add the Secondary Culture that micromolecular inhibitor PD0325901 and CHIR99021 carries out cell.This culture system is classical methods for production of mesodermal lineage cells, thus obtained cytotrophoblast stem cells is that the stem cell of originating with trophectoderm may exist the difference of some essence and potential feature by the embryonic stem cell derivative class cell be transformed under given conditions.
Summary of the invention
In order to overcome the problems referred to above of the prior art, the invention provides a kind of special culture media cultivating pig cytotrophoblast stem cells.
The special culture media of cultivation pig cytotrophoblast stem cells provided by the invention, composition comprises bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol.The all commercialization of above composition, directly can buy commercial goods, and such as wherein bFGF, activin A, Knockout SR and beta-mercaptoethanol can purchased from Gibco brands, and Y27632 can purchased from Sigma brand.
BFGF: Prostatropin is a kind of short fissional heparin-binding protein, in multiple biologically processes of fetal development and differentiation, activate FGF signal path, be a kind of in cell cultures widely used biological activity protein.
Activin A: activin A, the member of cytokine TGF beta superfamily, the signal path of TGF beta superfamily mediation there occurs different biological effects in maintenance embryonic stem cell self and special atomization, the stage of the main dependent cells received signal of the somatic destiny of tool, and the level of cytokine and the interaction with other cytokines.The independent long term of bFGF and activin A all can differentiation of stem cells, and can maintain the versatility of stem cell during their actings in conjunction, has implied the mechanism that there are regulation and control mutually between them.
Y27632: chemical formula C
14h
21n
3o, be a kind of Rho protein kinase (ROCK) specific inhibitor, structural formula is as follows:
There are some researches show that Y27632 can cause human embryo stem cell form to change, by quality closely cell colony become loose flat, Y27632 is applied to human embryo stem cell and goes down to posterity, can passaging efficiency be significantly improved.In the maintenance of embryonic stem cell characteristic, the action character of ROCK inhibitor is not also come to a conclusion, but in the present invention, by carrying out morphological observation evaluation and the detection of stem cell marker genes expression level to pig cytotrophoblast stem cells, prove that Y27632 can not maintain the versatility of stem cell to have a negative impact, and the apoptosis effect occurred in energy T suppression cell succeeding generations, illustrate that it can safely for the cultivation of pig cytotrophoblast stem cells.
Knockout SR:Knockout serum substitute is the serum-free preparation that composition is determined, for alternative foetal calf serum, promotes not break up multipotential stem cell growth.Traditional embryonic stem cell medium formula needs foetal calf serum mostly, containing the composition of many unknowns and the factor of some Cell differentiation inducing activities in serum, and can cause the pollution problem of foreign sera albumen.Therefore in order to address this problem, scientists proposes serum free culture system, develops Knockout SR and effectively can maintain the undifferentiated state of embryonic stem cell, maintains stem cells hyperplasia.
Beta-mercaptoethanol: beta-mercaptoethanol energy induced cell proliferation, promotes DNA synthesis, superoxide can be avoided the infringement of cultured cell in vitro simultaneously, hinder the differentiation due of multipotent stem cells.Be usually used in cultivating embryonic stem cell, induction versatile stem cell and neural stem cell etc.
Experiment finds that the combination of bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol effectively can maintain rapid propagation and the renewal of pig cytotrophoblast stem cells.In culture system, wherein remove bFGF, activin A or Knockout SR causes cell proliferation rate significantly to reduce, and does not add Y27632 and then causes cell major part after going down to posterity to occur apoptosis phenomenon, only have an appointment 30% the continued growth of cell energy.And stem cell clone edge fluffs loose after not adding beta-mercaptoethanol, cell trend differentiation state.
Preferably, in the special culture media of above-mentioned cultivation pig cytotrophoblast stem cells, the concentration of bFGF is 12.5 ~ 50ng/mL, the concentration of activin A is 5 ~ 20ng/mL, the concentration of Y27632 is 2 ~ 10nmol/mL, the volumetric concentration of Knockout SR is 2% ~ 5%, and the concentration of beta-mercaptoethanol is 20 ~ 55 nmol/mL.BFGF, activin A, Y27632, Knockout SR of this range of concentrations and beta-mercaptoethanol, versatility and the propagation that more effectively can maintain stem cell upgrade.
Preferably, in the special culture media of above-mentioned cultivation pig cytotrophoblast stem cells, DMEM/F12 substratum, Neurobasal substratum, N-2 supplement, B-27 supplement, MEM NEAA and L-glutaminate is also comprised.The all commercialization of above composition, can directly buy, and such as these compositions all can purchased from Gibco brand.
DMEM/F12 medium component is complicated, containing various trace elements, is applicable to the cultivation of serum content compared with mammalian cell under low condition.
Neurobasal substratum is the basic medium for meeting in utero first cell special requirement with fetal nerve, is applicable to the growth of the epithelium class cells such as neurocyte.
N-2 supplement is that chemical composition is determined, not containing the additive of serum, is mainly used in Neurobasal substratum.B-27 supplement is serum-free additive, can combinationally use with Neurobasal substratum.In culture system of the present invention, N-2 supplement and B-27 supplement collocation use can promote trophoderm epithelioid cell adherent growth.
MEM NEAA contains glycine, ALANINE, L-Aspartic acid, altheine, Pidolidone, L-PROLINE and Serine.This non-essential amino acid needed for 7 kinds of cell cultures can effectively improve cell culture medium proportioning, and when reducing cell cultures, cell self produces the side effect of non-essential amino acid.L-glutaminate is the indispensable amino acid of Growth of Cells, after desamidizate, for cultured cells provides important energy derive, and participates in synthesis and the nucleic acid metabolism of protein.In culture system of the present invention, add MEM NEAA and L-glutaminate be conducive to maintaining cells in vitro long-term cultivation.
Preferably, in the special culture media of above-mentioned cultivation pig cytotrophoblast stem cells, by DMEM/F12 substratum and Neurobasal substratum according to nutrient solution based on the volume ratio mixing of 1:1; In every 20mL basic culture solution, add N-2 supplement 50 ~ 200 μ L, B-27 supplement 100 ~ 400 μ L, MEM NEAA50 ~ 200 μ L, L-glutaminate 100 ~ 200 μ L.
Present invention also offers a kind of method of cultivating pig cytotrophoblast stem cells, comprise the following steps:
(1) stem cell culture vessel is in advance with extracellular matrix bag quilt;
(2) pig blastocyst protease digestion removes zona pellucida, proceeds to the stem cell culture vessel that step (1) is extracellular matrix coated, uses the special culture media of above arbitrary described cultivation pig cytotrophoblast stem cells to cultivate;
(3) be cultured to and form total cell count when being the cloning cluster of 1000 ~ 2000, be digested to unicellular, proceed to the Secondary Culture that the extracellular matrix coated culture vessel of new step (1) carries out cell.
Preferably, in the method for above-mentioned cultivation pig cytotrophoblast stem cells, described blastaea is blastaea, the blastaea in body-cell neucleus transplanting source, the blastaea in source in vitro fertilization or the lonely female blastaea that artificial insemination is originated.
Preferably, in the method for above-mentioned cultivation pig cytotrophoblast stem cells, the extracellular matrix in step (1) is rat collagen I, matrigel Matrigel or fibronectin.The commercialization all of above composition, can directly buy, and such as these compositions all can purchased from Gibco brand.
Rat collagen I is common fibrous collagen, and it is present in skin, bone, tendon and other reticular tissue, and the rat collagen I of Gibco brand can dilute neatly, is generated the gelatin of sclerosis by gelation step.
The matrigel Matrigel of Gibco brand is the basement membrane matrix extracted from the EHS mouse tumor being rich in extracellular matrix protein, its main component has ln, type Ⅳ collagen, nidogen, heparin sulfate glycoprotein, also comprises somatomedin and matrix metalloproteinase etc.At ambient temperature, Matrigel polymerization forms the three dimensional matrix with biologic activity.
Fibronectin is a kind of extracellular glycoprotein, is present in body fluid respectively with soluble form, or is present in extracellular matrix with soluble form.It can be interconnected with cell fiber, protein-polysaccharide, plays adhesive effect.
In the present invention, experimental verification uses rat collagen I, matrigel Matrigel or fibronectin bag by the culture vessel crossed, greatly can increase the adhesivity bottom cell culture vessel, impel pig blastocyst adherent, isolate cytotrophoblast stem cells, and when promoting that cell fission is bred and the attachment of culture dish, keep cell clone group adherent growth.
Preferably, in the method for above-mentioned cultivation pig cytotrophoblast stem cells, the proteolytic enzyme in step (2) is pronase; Blastaea before digestion first with Pig embryos nutrient solution PZM-3 cleaning, then proceed to cultivate pig cytotrophoblast stem cells special culture media in clean; Carry out the envrionment temperature 39 DEG C, the CO that cultivate
2volumetric concentration 5% and saturated humidity.
Preferably, in the method for above-mentioned cultivation pig cytotrophoblast stem cells, in step (3), the method for Secondary Culture is: after DPBS rinsing, add appropriate TRYPLE select trypsinase and substitute enzyme (Gibco brand) digestion for after unicellular, add pig cytotrophoblast stem cells special culture media nutrient solution stop digestion, piping and druming cell suspension, collect centrifugal after, with the special culture media dilution of cultivating pig cytotrophoblast stem cells, be passaged in the stem cell culture vessel with extracellular matrix bag quilt with the ratio of 1:3.
The present invention also provides the method for above-mentioned cultivation pig cytotrophoblast stem cells to cultivate the pig cytotrophoblast stem cells obtained.
Compared with prior art, the present invention has following beneficial effect:
Use feeder layer cells in conventional dry cell culture experiments method can secrete the somatomedin of some key, promotes the propagation of totipotent cell, and removes its differentiation of toxin and suppression in vitro culture conditions.But, the interference that a large amount of non-principal component identify stem cell and there is heterologous cells pollution problem all can influence research personnel to the research of stem cell culture system, so the feeder free system setting up cytotrophoblast stem cells has more advantage than there being feeder layer system.
The present invention successfully develops the feeder free system of livestock large animal cytotrophoblast stem cells first, replaces traditional allogeneic feeder confluent monolayer cells support by the combinations of. growth factors nutrient solution screening clear and definite chemical composition.This special culture media composition is determined, safe to use, avoid the pollution of heterologous cells, for the biological mechanism of such stem cell of research, and early embryo development and placenta differentiation significant, and the application that will advance cytotrophoblast stem cells further on somatic cell clone and large animal transgenic research.
The method of cultivation pig cytotrophoblast stem cells provided by the invention is simply efficient, cultivates have that cloning efficiency is high, apoptosis rate is low, propagation passage capacity is strong and the advantage of safety and stability to pig cytotrophoblast stem cells.Wherein artificial insemination source blastaea can form the efficiency of stem cell clone is 38.79%, and the efficiency that body-cell neucleus transplanting source blastaea can form stem cell clone is that 6.34%(statistics is as shown in table 1), and maintenance stem cell properties that can be steady in a long-term.
Accompanying drawing explanation
Fig. 1 is the form (A: the blastaea in artificial insemination source, B: the blastaea in body-cell neucleus transplanting source) of pig blastocyst.
Fig. 2 is the form (A: the blastaea in artificial insemination source, B: the blastaea in body-cell neucleus transplanting source) of the pig blastocyst after pronase digestion zona pellucida.
Fig. 3 is that pig blastocyst is inoculated in the special substratum of cytotrophoblast stem cells, and the form of adherent growth (A: the artificial insemination source blastaea inoculating latter second day, B: the body-cell neucleus transplanting source blastaea inoculating latter second day, C: the inoculation artificial insemination of latter 3rd day source blastaea adherent growth, D: the inoculation body-cell neucleus transplanting of latter 3rd day source blastaea adherent growth).
Fig. 4 is F0 generation and the growthhabit of the pig cytotrophoblast stem cells in F3 generation (the pig cytotrophoblast stem cells that A:F0 is separated for artificial insemination source blastaea, the pig cytotrophoblast stem cells that B:F3 is separated for artificial insemination source blastaea, the pig cytotrophoblast stem cells that C:F0 is separated for body-cell neucleus transplanting source blastaea, the pig cytotrophoblast stem cells that D:F3 is separated for body-cell neucleus transplanting source blastaea.
Fig. 5 is the expression that agarose gel electrophoresis detects the correlating markings gene of pig cytotrophoblast stem cells.AI-TSCs-cDNA is the pig cytotrophoblast stem cells cDNA that artificial insemination source blastaea is separated, SCNT-TSCs-cDNA is the pig cytotrophoblast stem cells cDNA that body-cell neucleus transplanting source blastaea is separated, Trophoblasts-cDNA is the trophocyte system cDNA broken up, and Fibroblasts-cDNA is the cDNA of fetal fibroblast cell line.
Fig. 6 is that immunofluorescent staining detects pig cytotrophoblast stem cells different expression gene CDX2 and Eomes(A and is classified as cellular form under white light, B shows hoest33342 to nuclear targeting under being classified as uv excitation light, C is classified as with CDX2 or Eomes antibody incubation and develops the color after adding fluorescence two anti-binding, and D is classified as the design sketch of B and C column weight poststack.Right side AI-TSCs represents that the pig cytotrophoblast stem cells that artificial insemination source blastaea is separated, SCNT-TSCs represent the pig cytotrophoblast stem cells that body-cell neucleus transplanting source blastaea is separated.
Fig. 7 be pig cytotrophoblast stem cells differentiation-inducing after cellular form (A: artificial insemination source blastaea be separated cytotrophoblast stem cells differentiation after form polykaryocyte, B: form trophocyte after the cytotrophoblast stem cells differentiation that artificial insemination source blastaea is separated, C: form polykaryocyte, D after the cytotrophoblast stem cells differentiation that body-cell neucleus transplanting source blastaea is separated: form trophocyte after the cytotrophoblast stem cells differentiation that body-cell neucleus transplanting source blastaea is separated.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but is not used for limiting practical range of the present invention.If do not specialize, the conventional means that technique means used in embodiment is well known to the skilled person, is raw materials usedly commercial goods.
Embodiment one: the pig cytotrophoblast stem cells separation and Culture of artificial insemination (Artificial Fertilizing, AI) blastaea of originating and qualification
1. the foundation of the culture system of the pig cytotrophoblast stem cells of AI source blastaea
The acquisition methods of 1.1 blastaeas:
Adopt the method for AI to breed to the common sow that oestruses, adopt the method for rushing embryo in vitro to collect blastaea afterwards in breeding.Concrete operations are: select the sow of high gestational age butchering for the 6th day or the 7th day after breeding, take out uterus, horn of uterus front end is fixed with mosquito forceps, egg liquid (DPBS+PVA) is injected from fimbriae tubae portion, collect in horn of uterus position with conduit, filter cleaning by egg liquid, observe and collect blastaea under stereoscopic microscope, 39 DEG C of constant temperature are transported to laboratory (artificial insemination source blastaea form is as Figure 1A).
The special culture media of 1.2 preparation pig cytotrophoblast stem cells:
By DMEM/F12 and Neurobasal substratum according to nutrient solution based on the volume ratio mixing of 1:1;
In every 20mL basic culture solution, add N-2 supplement 50 ~ 200 μ L, B-27 supplement 100 ~ 400 μ L, MEM NEAA 50 ~ 200 μ L, L-glutaminate 100 ~ 200 μ L.And concentration is 12.5 ~ 50ng/mL bFGF, 5 ~ 20ng/mL activin A, 2 ~ 10nmol/mL Y27632,20 ~ 55nmol/mL beta-mercaptoethanol and volumetric concentration are the Knockout SR of 2% ~ 5%.
The separation of 1.3 primary cytotrophoblast stem cells and cultural method
1.3.1 extracellular matrix coated cell culture vessel
Can select from following A, B and C tri-kinds of methods wherein any one, action effect is similar.
A: rat collagen I bag is by culture dish: specific experiment step, for diluting rat I Collagen Type VI to 50 ~ 200 μ g/mL by 0.02 mole of acetic acid, then presses 5 ~ 20 μ g/cm
2add to Nostoc commune Vanch ware (Corning brand), after at room temperature hatching 1 hour, careful suction is abandoned, and cleans three times, dry for subsequent use with DPBS.
B: matrigel Matrigel bag is by culture dish: specific experiment step is with DMEM/F12 the concentration dilution to 50 of Matrigel ~ 200 μ g/mL, then by 5 ~ 20 μ g/cm
2add to Nostoc commune Vanch ware (Corning brand), hatch 1 hour at 37 DEG C after, careful suction is abandoned, and dries for subsequent use.
C: fibronectin bag is by culture dish: specific experiment step, for fibronectin is diluted to 50 ~ 200 μ g/mL with DMEM/F12, then presses 5 ~ 20 μ g/cm
2add to Nostoc commune Vanch ware (Corning brand), after at room temperature hatching 1 hour, careful suction is abandoned, and dries for subsequent use.
1.3.2 inoculate blastaea and primary stem cell clone to be formed: the pig blastocyst embryo medium PZM-3 being developed to the 6th day or the 7th day cleans twice, zona pellucida (removing the embryo morphology after zona pellucida as Fig. 2 A) is removed again with pronase digestion, proceed in stem cell media and clean twice, then the blastaea that complete for zona pellucida cracking disappears is sucked in special culture media of the present invention.Proceed to 39 DEG C, 5%CO
2cultivate (within second day, observing postvaccinal blastaea form as Fig. 3 A) with in the incubator of saturated humidity environment, within the 3rd day, observe the adherent and cell clone growthhabit (the adherent formation colony morphology of stem cell is as Fig. 3 C) of blastaea.The final efficiency that artificial insemination source blastaea is separated cytotrophoblast stem cells system is 38.79%, and specific experiment data are as following table 1.
Table 1
Going down to posterity and cryopreservation methods of 1.4 pig cytotrophoblast stem cells
Propagating method used is enzyme digestion, namely intensive in stem cell clone group, when there is contact inhibition in cell, with DPBS rinsing twice, add appropriate TRYPLE select trypsinase and substitute enzyme (Gibco brand) digestion for unicellular, add pig cytotrophoblast stem cells special culture media and stop digestion, cell suspension is blown and beaten with liquid transfer gun head, collect centrifugal after, be passaged to new extracellular matrix bag by (F0 generation and F3 for stem cell morphology respectively as Fig. 4 A and Fig. 4 B) in the culture dish crossed with the ratio of 1:3.The cryopreservation methods of pig cytotrophoblast stem cells is peptic cell, after centrifugal, add commercial freezing media (Gibco brand), even with liquid transfer gun head piping and druming cell suspension, be transferred to (corning brand) in cryopreservation tube, be placed in 4 DEG C and preserve 30 minutes, preserve 30 minutes in negative 20 DEG C afterwards, in negative 80 DEG C, preserve 16-48 hour afterwards, be transferred to the medium-term and long-term preservation of liquid nitrogen afterwards.
2. the marker gene expression identification of pig cytotrophoblast stem cells
2.1 RT-PCR technology gene expression detection situations
Detection comprises cytotrophoblast stem cells specific expression gene Eomes, Cdx2 and Tead4; Versatility gene Oct4, Nanog and Sox2; Trophoderm specific expression gene Cyp17a1, Plet, Krt8 and Hand1; Fibroblast growth factor acceptor 2 gene Fgfr2, x chromosome inactivation genes involved Xist; And reference gene Gapdh.
Specific experiment step: clean with PBS after the digestion of step 1 cultured cells, the RNA extraction agent box of Qiagen company is adopted to extract RNA, carrying out reverse transcription with the Reverse Transcription box of Fermentas company is that cDNA preserves, above each gene order design Auele Specific Primer for pig increases, whether agarose gel electrophoresis detects gene and expresses, the cDNA amplification of trophocyte system and the fetal fibroblast cell line broken up is employed in contrast in experiment, result shows the cytotrophoblast stem cells high expression level Eomes that artificial insemination is originated, Cdx2, Tead4, Cyp17a1, Plet, Krt8, Hand1 and Fgfr2, weak expression Oct4, Nanog and Sox2(electrophoresis detection stem cell gene expression as shown in Figure 5).
Detect all genetic expression PCR primer sequence used as following table 2:
Table 2
2.2 immunofluorescent staining methods detect cytotrophoblast stem cells different expression gene CDX2 and Eomes
The pig cytotrophoblast stem cells in the F3 generation that special culture media of the present invention is cultivated is when growing to degree of converging about 80%, with DPBS cleaning twice, add 4% paraformaldehyde and fix 5 minutes, bored a hole by TritonX-100 cell membrane afterwards, then add lowlenthal serum and close 30 minutes, add again 1:200 dilution after CDX2 or Eomes primary antibodie overnight incubation, clean more than three times with DPBS, afterwards lucifuge add 1:1000 dilution after Alexa Fluor
?568 fluorescent marks two resist, display red fluorescence.DAPI nuclear staining afterwards, mounting, microscopy, takes pictures under inverted fluorescence microscope, and result display CDX2 albumen equal great expression in tenuigenin and nucleus, Eomes albumen is great expression (expression of CDX2 and Eomes as shown in Figure 6) in nucleus.
3. the Multidirectional Differentiation Function Identification of pig cytotrophoblast stem cells
In 3.1 experiments, the inducing culture designed to polykaryocyte differentiation is 90%DMEM/F12 and 10% foetal calf serum (Gibco brand).Specific experiment step is remove stem cell medium after F3 cultivates 24 hours for cytotrophoblast stem cells, with DPBS cleaning twice, is replaced by division culture medium and continues to cultivate.Liquid is changed every two to three days.
Find in experiment to cultivate after three days in division culture medium, the cell of about 5% has started differentiation becomes polykaryocyte, and after successive induction breaks up 15 days, the cell of about 50% has broken up becomes typical polykaryocyte (polykaryocyte form as shown in Figure 7 A).
The inducing culture that in 3.2 experiments to design to trophocyte be differentiation is 95%DMEM/F12,5% foetal calf serum FBS and 0.1Unit/mL Sigma I8405.Specific experiment step is remove stem cell medium after F3 cultivates 24 hours for cytotrophoblast stem cells, with DPBS cleaning twice, is replaced by above inducing culture and continues to cultivate.Liquid is changed every two to three days.Find in experiment to cultivate after the week in inducing culture, cell clone is loose, and the speed of growth is slow, and lamellar growth, in flat characteristics of epithelial cells (morphology of trophoblast cells after differentiation is as shown in Fig. 7 B).
Embodiment two: the pig cytotrophoblast stem cells separation and Culture in body-cell neucleus transplanting (Somatic cell nuclear transfer, SCNT) blastaea source and qualification
1. the foundation of the culture system of the pig cytotrophoblast stem cells of SCNT blastaea source separation
The acquisition of 1.1 SCNT blastaeas: adopt micrurgy instrument to carry out the nuclear transplantation of pig inoblast and obtain clone embryos, the acquisition of porcine oocytes and maturation culture, for the fibroblastic cultivation of core, the stoning of ovocyte and note core, ovocyte and somatic fusion and activate and all operate (Li et al. according to existing documentation standards, 2013, PMID:24033142) embryo medium PZM-3 cultivate reconstructed embryo to the 6th day or the 7th day, collect blastaea.Originate compared with blastaea with AI, its small volume, in blastaea, total cell number is less, embryo quality poor (SCNT blastaea form is as Figure 1B).
1.2 SCNT the originate establishment method of culture system of pig cytotrophoblast stem cells of blastaea of all the other establishment method and the AI described in embodiment one of culture system of pig cytotrophoblast stem cells of blastaeas of originating is consistent, and the final efficiency of SCNT blastaea separation cytotrophoblast stem cells system is 6.34%.(remove the embryo morphology after zona pellucida as Fig. 2 B, within second day, observe postvaccinal blastaea form as Fig. 3 B, the adherent formation colony morphology of stem cell in SCNT source is as Fig. 3 D, the stem cell morphology that F0 generation and F3 originate for SCNT is respectively as Fig. 4 C and Fig. 4 D, SCNT blastaea are separated the specific experiment data of the efficiency of cytotrophoblast stem cells system as table 1).
2. the correlating markings gene identification of the cytotrophoblast stem cells in SCNT source
The correlating markings gene identification method consistent with method described in embodiment one (expression conditions of SCNT Derived Stem Cells as shown in Figure 5) of the cytotrophoblast stem cells in 2.1 SCNT sources.
2.2 immunofluorescent staining methods detect cytotrophoblast stem cells different expression gene CDX2 with Eomes: basic skills is consistent with embodiment one, change adopt two resist for Alexa Fluor 488 mark fluorescence antibody, display green fluorescence (expression of CDX2 and Eomes as shown in Figure 6).
3. the Multidirectional Differentiation Function Identification method of cytotrophoblast stem cells in SCNT source consistent with described in embodiment one (SCNT originate cytotrophoblast stem cells break up after polykaryocyte form as seen in figure 7 c, the morphology of trophoblast cells after differentiation is as illustrated in fig. 7d).
The present invention belongs to the culture system without feeder layer cells successfully developing livestock large animal cytotrophoblast stem cells first at home and abroad, traditional allogeneic feeder layer support is replaced by the combinations of. growth factors nutrient solution screening clear and definite chemical composition, avoid the pollution of heterologous cells, for the biological mechanism of such stem cell of research, and early embryo development and placenta differentiation significant, and the application that will advance cytotrophoblast stem cells further on somatic cell clone and large animal transgenic research.
Claims (10)
1. cultivate a special culture media for pig cytotrophoblast stem cells, it is characterized in that, composition comprises bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol.
2. the special culture media of cultivation pig cytotrophoblast stem cells according to claim 1, it is characterized in that, the concentration of bFGF is 12.5 ~ 50ng/mL, the concentration of activin A is 5 ~ 20ng/mL, the concentration of Y27632 is 2 ~ 10nmol/mL, the volumetric concentration of Knockout SR is 2% ~ 5%, and the concentration of beta-mercaptoethanol is 20 ~ 55 nmol/mL.
3. the special culture media of cultivation pig cytotrophoblast stem cells according to claim 1 and 2, it is characterized in that, also comprise DMEM/F12 substratum, Neurobasal substratum, N-2 supplement, B-27 supplement, MEM NEAA and L-glutaminate.
4. the special culture media of cultivation pig cytotrophoblast stem cells according to claim 3, is characterized in that,
By DMEM/F12 substratum and Neurobasal substratum according to nutrient solution based on the volume ratio mixing of 1:1;
In every 20mL basic culture solution, add N-2 supplement 50 ~ 200 μ L, B-27 supplement 100 ~ 400 μ L, MEM NEAA 50 ~ 200 μ L, L-glutaminate 100 ~ 200 μ L.
5. cultivate a method for pig cytotrophoblast stem cells, it is characterized in that, comprise the following steps:
(1) stem cell culture vessel is in advance with extracellular matrix bag quilt;
(2) pig blastocyst protease digestion removes zona pellucida, proceeds to the stem cell culture vessel that step (1) is extracellular matrix coated, uses the special culture media of the arbitrary described cultivation pig cytotrophoblast stem cells of claim 1 ~ 4 to cultivate;
(3) be cultured to and form total cell count when being the cloning cluster of 1000 ~ 2000, be digested to unicellular, proceed to the Secondary Culture that the extracellular matrix coated culture vessel of new step (1) carries out cell.
6. the method for cultivation pig cytotrophoblast stem cells according to claim 5, is characterized in that, described blastaea is blastaea, the blastaea in body-cell neucleus transplanting source, the blastaea in source in vitro fertilization or the lonely female blastaea that artificial insemination is originated.
7. the method for cultivation pig cytotrophoblast stem cells according to claim 5, it is characterized in that, the extracellular matrix in step (1) is rat collagen I, matrigel Matrigel or fibronectin.
8. the method for cultivation pig cytotrophoblast stem cells according to claim 5, it is characterized in that, the proteolytic enzyme in step (2) is pronase; Blastaea before digestion first with Pig embryos nutrient solution PZM-3 cleaning, then proceed to cultivate pig cytotrophoblast stem cells special culture media in clean; Carry out the envrionment temperature 39 DEG C, the CO that cultivate
2volumetric concentration 5% and saturated humidity.
9. the method for cultivation pig cytotrophoblast stem cells according to claim 5, it is characterized in that, in step (3), the method for Secondary Culture is: use DPBS rinsing, add appropriate TRYPLE select trypsinase substitute enzymic digestion be unicellular after, add pig cytotrophoblast stem cells special culture media stop digestion, piping and druming cell suspension, collect centrifugal after, with the special culture media dilution of cultivating pig cytotrophoblast stem cells, be passaged in the stem cell culture vessel with extracellular matrix bag quilt with the ratio of 1:3.
10. the method for the cultivation pig cytotrophoblast stem cells described in claim 5 ~ 9 cultivates the pig cytotrophoblast stem cells obtained.
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