CN104694471A - Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro - Google Patents
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Abstract
The invention discloses an effective method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro. The embryonic stem cells are pre-induced through an inducing system containing dexamethasone, subjected to induction culture through a serum-free medium system containing a serum-free culture additive N2 or B27, prostaglandin E2 and L-glutamine, and then induced to be differentiated into the erythroid cells through a serum-free medium system containing stem cell factors (SCF), interleukin 3 and hemopoietin. The method specifically includes the following steps of conventionally culturing the embryonic stem cells in vitro, pre-inducing the embryonic stem cells, directionally differentiating the pre-induced cells into hematopoietic stem cells, and directionally differentiating the hematopoietic stem cells into the erythroid cells. According to the method for inducing the embryonic stem cells to be differentiated into the erythroid cells in vitro, materials are convenient to obtain, expansion in vitro is facilitated, immunogenicity is low, and therefore the human embryonic stem cells are differentiated into the mature erythroid cells more safely and efficiently, and the technological application prospects are good.
Description
Technical field
The invention belongs to regenerative medicine and biological technical field, relate to the preparation method of erythroid cells, especially a kind of external evoked embryonic stem cell is to the method for erythroid diffrentiation.
Background technology
That lives require along with the fast development of China's medical skill and people increasing day by day, and component blood transfusion deepens continuously in clinical treatment practice, and wherein the consumption of erythroid cells (also claiming hemocyte) is maximum.Erythroid cells infusion has been vital cell therapy means in modern medical service technology, and object mainly improves the anoxic condition of patient, is widely used in postoperative and wound, chronic anaemia and other various diseases treatment.But still relying on this approach of volunteer's voluntary blood donation clinically is at present major blood source, and blood supply is at full stretch, and the report of blood supply deficiency is through being common in the various media such as newspaper, TV and network.Updating along with detection technique at present, transfusional transmissible disease incidence is very low, but still happens occasionally.The security that these problems are applied clinical blood transfusion and popularity bring stern challenge, therefore the important directions that safer and economic blood sources is blood transfusion medical research is always found, people have turned one's attention to produced in vitro erythroid cells, the external preparation of erythroid cells can produce specific blood group on demand, and the generation etc. of relevant communicate illness of can avoiding to greatest extent transfusing blood.
Develop multiple erythroid cells surrogate at present, mainly comprise hemoglobin-based oxygen carrier and the large class of fluorine carbon compound two.But hemoglobin-based oxygen carrier poor stability, purification difficult, complex process and cost are high, fluorine carbon compound oxygen carrying capacity is weak, metabolism is slow, and there is very large toxic side effect, therefore this two classes erythroid cells substitute still can not be applied on a large scale, needs to continue to find new blood erythroid cells and substitutes source.Stem cell and application thereof has in recent years become one of focus of world's life science, is that the fields such as the functional study of correlative study at cell therapy, fetal development, new gene of technology platform, gene therapy, drug screening and new drug pharmacological research all show tempting application prospect with stem cell.
Stem cell has self and Multidirectional Differentiation ability in vitro, its exclusive characteristic: the 1) self-renewal capacity of height, has close to unlimited multiplication capacity in vitro; 2) totipotency of Development And Differentiation; 3) operability of heredity, makes its seed cell as Differentiation Induction in vitro have obvious advantage.Cell therapy based on human embryo stem cell vitro directed differentiation may bring hope for the treatment of a lot of disease, wherein human embryo stem cell is induced to differentiate in vitro on a large scale mature erythrocyte and can be used as the alternative source of new blood likely for solution current clinical blood transfusion treatment problems faced brings hope, will be with a wide range of applications and great economic results in society.
At present, inducing in vitro human ES cell differentiation is ripe erythroid cells and is finally applied to clinical a lot of crucial technical problem in addition to need to solve, comprise being broken up to hemopoietic stem cell by human embryo stem cell of how safe low cost, how high-level efficiency inducing hematopoietic stem cell is to erythroid diffrentiation, how to ensure whether the erythroid cells of differentiation-inducing maturation can play function in vivo, induction obtains the problem such as erythrocytic security and immunological rejection.In above problem, it is the external preparation method that active cell directional induction generates erythroid cells that the present invention sets up first with embryonic stem cell, intend by solving wherein two crucial technical problem, namely realize in vitro safe low cost by human embryo stem cell to hemopoietic stem cell break up and high efficiency under external serum free culture system be erythroid cells by hemopoietic stem cell directed differentiation.Wish to make human embryo stem cell can be safer, efficient to mature erythrocyte differentiation, for finally carrying out tachnical storage for obtaining by embryonic stem cell the erythroid cells meeting clinical blood transfusion needs by solving above two key technical problems.
Summary of the invention
The object of the invention is, provide a kind of and draw materials conveniently, be easy to the method for the low external evoked embryonic stem cell of amplification in vitro, immunogenicity to erythroid diffrentiation.
In order to solve the problems of the technologies described above, reach above-mentioned technique effect, the present invention realizes by the following technical solutions:
External evoked ES cell differentiation of the present invention is the method for erythroid cells, the induction system containing dexamethasone is adopted to carry out pre-induced to embryonic stem cell, and then adopt the serum free medium system inducing culture containing serum-free culture additive N2 or B27, prostaglandin E2 and L-glutaminate, then adopt the differentiation-inducing one-tenth erythroid cells of serum free medium system containing STEM CELL FACTOR (SCF), interleukin-13, erythropoietin.
The erythroid cells of the inventive method differentiation be a kind of for losing blood, ischemic, fluid loss, need the source supplementing the necessary colloidal solution of human body in time, by human embryo stem cell in vitro directed differentiation formed, and substitute blood products by the method for venoclysis.
The erythroid cells surface marker CD36 positive expression rate that the inventive method is differentiated to form is more than 90%.
Further, described external evoked ES cell differentiation is the method for erythroid cells, it is characterized in that being made up of following step:
(1) the external cellar culture of embryonic stem cell;
(2) pre-induced of embryonic stem cell: adopt by induction system I cultivation be prepared from containing dual anti-DMEM/F-12 perfect medium that with the addition of 0.1mM dexamethasone;
(3) cell directional of pre-induced is divided into hemopoietic stem cell: adopt comprise volume ratio concentration be 2% N2 or B27, containing dual anti-serum-free StemSpan ACF substratum, and with the addition of 20ng/ml prostaglandin E2, the L-glutaminate of 2mM is prepared from induction system II cultivation;
(4) hemopoietic stem cell directed differentiation becomes erythroid cells: adopt comprise volume ratio concentration be 2% N2 or B27, containing dual anti-serum-free StemSpan ACF substratum, and with the addition of 0.1mM STEM CELL FACTOR (SCF), induction system III cultivation that 0.15mM interleukin-13,0.05mM erythropoietin are prepared from.
Further, described step (1) specifically comprises the following steps: cellar culture embryonic stem cell carries out going down to posterity and increasing, this stage substratum is: substratum based on 85%DMEM substratum, add 15% Knockout Serum Replacement, 1mmol/L indispensable amino acid, 100IU/mL penicillin, 50 μ g/mL Streptomycin sulphates and 4ng/mL Prostatropin.Cell cultures, at 37 DEG C, is hatched under the carbon dioxide conditions of 5%, goes down to posterity weekly once.
Further, described step (2) specifically comprises the following steps: in order to inducing human embryo stem cell is to the differentiation of hematopoietic cell, within 6 ~ 7 days, induces embryonic stem cell in cultivation.At 37 DEG C, digest 3 ~ 5min with 1mg/mL type Ⅳ collagenase and blow and beat into small cell cluster, then ultralow 6 well culture plates that stick are used to suspend 7 days, 1st ~ 5 days adopt containing volume ratio concentration is 100IU/mL penicillin, the DMEM/F-12 complete culture solution of 50 μ g/mL Streptomycin sulphates is cultivated, within every 2 days, change liquid once, adopted at the 5th day that cultivates the induction system I adding pre-induced factor 0.1mM dexamethasone to cultivate 2 ~ 3 days, be placed in 37 DEG C, cultivate in 5% CO2gas incubator.
Further, described step (3) specifically comprises the following steps: 96 hole tissue culturing plates above-mentioned steps (2) pre-induced cultured cells being moved to 0.1% gelatin bag quilt.Adopt induction system II, the allos may brought because adding serum can be avoided to pollute, and put 37 DEG C, cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 2 days once; Within every 7 days, change liquid once, after adherent culture, every day observes cell growth status in culture dish under an optical microscope, cultivates and obtains the hemopoietic stem cell become by Oriented Differentiation of Embryonic Stem Cell afterwards in 20 days.
Further, described step (4) specifically comprises the following steps:
The first step, prepares stem cell by above-mentioned steps (3) and carries out through flow cytometer detection, and CD34 positive cell ratio is more than 90%, meets next step carries out amplification cultivation requirement to it;
Second step, successfully isolate CD34 positive cell by immunomagnetic beads method to be inoculated in culture dish and to cultivate, add 0.25% trypsinase-EDTA Digestive system 1.0ml, immerse and cover at the bottom of bottle, observe when seeing the rounded levitating of cell under inverted microscope, add 1.0ml foetal calf serum and stop digestion;
3rd step, adds 10ml PBS and repeatedly blows and beats flushing, at room temperature 900 revs/min centrifugal 10 minutes; After abandoning supernatant, add 10ml serum-free induction system III re-suspended cell, renewed vaccination is in culture dish;
4th step, puts 37 DEG C by above-mentioned culture dish, and cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 3 days once; Within every 7 days, change liquid once, cultivate the erythroid cells obtaining afterwards for 20 days and come by the thin directed differentiation of Hematopoietic Stem, collect erythroid cells and carry out flow cytometer detection, result display CD36 positive expression rate is more than 90%.
Compared with prior art, the invention has the beneficial effects as follows:
Induction method of the present invention realizes external by embryonic stem cell inductive formation hemopoietic stem cell first, is then carrying out and arriving generating erythroid cells.The hemopoietic stem cell prepared by the inventive method and erythroid cells specifically expressing high, hemopoietic stem cell CD 34 positive expression rate rises to 90% by 85% of prior art, and erythroid cells CD36 positive expression rate rises to more than 90% by 86% of prior art; And to set up first with embryonic stem cell be that active cell directional induction generates the external preparation method of erythroid cells, there is stronger creativeness.
The inventive method further provides abundance, safe, effective and economic cell derived, is more conducive to expanding blood infusion application; And the hemopoietic stem cell of middle inductive formation has the ability to red corpuscle and platelet cell differentiation equally, for the source of hematologic disease a kind of new hemopoietic stem cell as leukemic treatment also provides; Doing that induction uses carefully derives from embryonic stem cell, draws materials conveniently, is easy to amplification in vitro, immunogenicity is low.Because physiologic characteristic is basically identical, so the human stem cells no matter which kind of is originated, be all suitable for induction method provided by the present invention.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
The technical scheme implementing procedure figure of Fig. 1 to be external evoked ES cell differentiation be erythroid cells;
Fig. 2 adds the prostaglandin E2 of different concns and L-glutaminate to cultivate hematopoiesis erythrocytic 14d Colony forming ability and compare, and wherein: same letter or do not mark and represent that group difference is remarkable in figure, different letter representation group difference significantly or extremely remarkable;
Fig. 3 be hemopoietic stem cell specific to erythroid differentiation figure, Rui Shi-Giemsa staining cell at cultivation the 1st day (figure A), 6 days (figure B), 10 days (figure C), 12 days (figure D) growing states;
Fig. 4 is that CFU-E is inoculated in methylcellulose gum semisolid medium and cultivates 7 days BFU-E and CFU-E Colony forming (A, scale=100 μm; B, scale=50 μm.)；
Fig. 5 is the lower polychromatophilic erythroblast (hollow black arrow) of Rui Shi-Ji's nurse Sa dyeing and acidophilic normoblast (filled black arrows) form, original amplification: 1000 ×.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
Shown in the technical scheme implementing procedure figure that the external evoked ES cell differentiation of reference Fig. 1 is erythroid cells, the specific embodiment of the invention is as follows:
Specific embodiment 1: external evoked ES cell differentiation is erythroid cells
(1) cellar culture of embryonic stem cell, concrete steps comprise:
Cellar culture embryonic stem cell carries out going down to posterity and increasing, this stage substratum is: substratum based on 85%DMEM substratum, adds 15% Knockout Serum Replacement, 1mmol/L indispensable amino acid, 100IU/mL penicillin, 50 μ g/mL Streptomycin sulphates and 4ng/mL bFGF.Cell cultures, at 37 DEG C, is hatched under the carbon dioxide conditions of 5%, goes down to posterity weekly once.
(2) pre-induced of embryonic stem cell, concrete steps comprise:
In order to inducing human embryo stem cell is to the differentiation of hematopoietic cell, embryonic stem cell was induced in 6 ~ 7 days in cultivation.At 37 DEG C, digest 3 ~ 5min with 1mg/mL type Ⅳ collagenase and blow and beat into small cell cluster, then ultralow 6 well culture plates that stick are used to suspend 7 days, within 1st ~ 5 days, adopt containing 100IU/mL penicillin, the DMEM/F-12 complete culture solution of 50 μ g/mL Streptomycin sulphates is cultivated, within every 2 days, change liquid once, adopted at the 5th day that cultivates the induction system I adding pre-induced factor 0.1mM dexamethasone to cultivate 2 ~ 3 days, be placed in 37 DEG C, cultivate in 5% CO2gas incubator.
(3) stem cell directional of pre-induced is divided into hemopoietic stem cell, specifically expressing CD34+, and concrete steps comprise:
The stem cell that above-mentioned steps (2) pre-induced is cultivated is moved to 96 hole tissue culturing plates of 0.1% gelatin bag quilt.Adopt induction system II, the allos may brought because adding serum can be avoided to pollute, and put 37 DEG C, cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 2 days once; Within every 7 days, change liquid once, after adherent culture, every day observes cell growth status in culture dish under an optical microscope, cultivates and obtains the hemopoietic stem cell become by Oriented Differentiation of Embryonic Stem Cell afterwards in 20 days.Induction system II by comprise volume ratio concentration be 2% N2 or B27, containing dual anti-serum-free StemSpan ACF substratum, and with the addition of 20ng/ml prostaglandin E2, the L-glutaminate of 2mM is prepared from.
(4) hemopoietic stem cell directed differentiation becomes erythroid cells, specifically expressing CD36+, and concrete steps comprise:
The first step, prepares stem cell by above-mentioned steps (3) and carries out through flow cytometer detection, and CD34 positive cell ratio is more than 90%, meets next step carries out amplification cultivation requirement to it;
Second step, successfully isolate CD34 positive cell by immunomagnetic beads method to be inoculated in culture dish and to cultivate, add 0.25% trypsinase-EDTA Digestive system 1.0ml, immerse and cover at the bottom of bottle, observe when seeing the rounded levitating of cell under inverted microscope, add 1.0ml foetal calf serum and stop digestion;
3rd step, adds 10ml PBS and repeatedly blows and beats flushing, at room temperature 900 revs/min centrifugal 10 minutes; After abandoning supernatant, add 10ml serum-free induction system III re-suspended cell, renewed vaccination is in culture dish;
4th step, puts 37 DEG C by above-mentioned culture dish, and cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 3 days once; Within every 7 days, change liquid once, cultivate the erythroid cells obtaining afterwards for 20 days and come by the thin directed differentiation of Hematopoietic Stem, collect erythroid cells and carry out flow cytometer detection, result display CD36 positive expression rate is 93%.
Induction system III train by adopt comprise volume ratio concentration be 2% N2 or B27, containing 100IU/mL penicillin, the serum-free StemSpan ACF substratum of 50 μ g/mL Streptomycin sulphates, and with the addition of 0.1mM STEM CELL FACTOR (SCF), 0.15mM interleukin-13,0.05mM erythropoietin be prepared from.
Specific embodiment 2: the mensuration of hemopoietic stem cell indices
(1) cell surface specifically expressing: hemopoietic stem cell specificity marker detects
The glycoprotein of CD34 antigen to be cell surface molecule amount be 115kD, the gene of coding CD34 be positioned at No. 1 chromosomal long-armed, containing 8 exons.CD34 antigen is the cluster of differentiation antigen on the surface existing only in hematopoietic stem/progenitor.CD34 strong positive on hemopoietic stem cell is expressed, and is then the weak positive to breaking up compared with the progenitor cell in late period.Mature blood cell does not express CD34 antigen.Therefore, CD34 can be used as the basic sign detecting hemopoietic stem cell.
Fetch the cell of turning out Growth of Cells good from embodiment 1 step (3) to detect: collect, the centrifugal 10min of 1500rpm, abandons supernatant, add PBS solution resuspended, draws 100 μ L cell suspensions (about 3 × 10
5individual cell) add in EP pipe, add detection antibody respectively according to quantity; Lucifuge hatches 30min, and every sample hose adds 2mlPBS and washes once, the centrifugal 5min of 1500rpm; Abandon supernatant, it is resuspended that every sample hose adds 300 μ LPBS solution, carries out flow cytometer detection.As follows through flow cytomery result: negative control: 0.07%; Positive mark's thing is expressed: CD34 is 90.35%; Negative marker thing is expressed: CD38 is 1.81%, CD44 is 0.01%.
(2) the Colony forming ability of inducing cell detects
Collect the cell of directional induction 20 ~ 22d in embodiment 1 step (3), be resuspended in 300 μ lPBS liquid, add non-specific blocking antibody FcR encapsulant 100 μ l, mix, then add the CD34 monoclonal antibody 100 μ l of magnetic bead coupling, mix, 2 times are washed with PBS liquid after hatching 30min in 4 DEG C, 1800r/min, centrifugal 5min, and then resuspended rear for subsequent use with PBS liquid.Separator column is placed in magnetic field, rinses separator column with PBS liquid, the cell suspension of mark is slowly added separator column, after it flows out naturally, washs uncombined cell with 500 μ l PBS liquid again.Finally separator column is withdrawn from magnetic field, add the cell of PBS liquid 1ml pressurization wash-out separator column absorption, collect the cell be separated and be CD34+ cell.
Embodiment 1 step (3) is trained for a certain area and is inoculated in 20ml semisolid medium by the isolated cell of magnetic bead coupling, semisolid medium moiety refers to as follows: 0.9% methylcellulose gum, 25%IMDM, 5%PFHM-II, 15% foetal calf serum, 1%BSA, 1mM is dual anti-, 2mM L-glutaminate, 200 μ g/ml Transferrins,iron complexess, 2 mM Sodium.alpha.-ketopropionates, MTG(stoste 1:100 dilutes), the prostaglandin E2 (PGE2) and the L-glutaminate that add different concns detect its Colony forming ability, that A group does not add PGE2 and L-glutaminate for control group respectively, B group adds the L-glutaminate of PGE2 and 1mM of 10ng/ml, C group adds the L-glutaminate of PGE2 and 2mM of 20ng/ml, C group adds the L-glutaminate of PGE2 and 3mM of 30ng/ml, D group adds the L-glutaminate of PGE2 and 4mM of 40ng/ml.
Illustrate shown in middle Fig. 2 the results detailed in accompanying drawing, the formation of hematopoietic colonies can be observed in semisolid medium after cultivating 6d, along with the quantity of the prolongation colony of incubation time increases gradually, and reach peak value in the 14d cultivated, now under inverted microscope, statistical counting is carried out to the hematopoietic colonies that each group of cell generates.The PGE2 of 100ng/ml can suppress the growth of colony, and the PGE2 of 20ng/ml has obvious promoter action to colony growth, and the ability that the cell that induction produces forms colony is the strongest, and the hematopoietic colonies of generation is maximum.
Specific embodiment 3: the indices of erythroid cells measures
1, erythroid cells smear test
(1) cell of embodiment 1 step (4) different time points suspension culture is collected, 2000 revs/min, centrifugal 5 minutes.Abandon supernatant, add 1mlPBS and clean cell twice.By resuspended for cell to 100 μ l;
(2) slide glass is through acid soak, and distilled water is rinsing repeatedly, with poly-lysine bag quilt.Be installed on cell slide machine according to operation instructions, use pencil to carry out mark;
(3) add cell suspension, setting cell centrifugation smearing machine rotating speed is 1000 revs/min, centrifugal 2 minutes.Centrifugal complete, middle filter paper layer and slide glass are prolonged vertical direction and is separated, carefully take off slide glass, slide glass is dried.
Result shows, and hemopoietic stem cell is in the also amplification rapidly of red system inducing culture suspension growth, and hemopoietic stem cell is inoculated in and with the addition of in the red system inductive differentiation medium of inducible factor, cell is suspension growth, propagation is very fast, and within 2 ~ 3 days, go down to posterity once, the ratio of going down to posterity is 1:3 ~ 1:6.At the beginning of separation, cellular form is comparatively homogeneous, spherical in shape, and along with the prolongation of induction time, size is uneven because propagation degree is different for cell.
2, erythroid cells Rui Shi-Ji's nurse Sa stain test
(1) by slide glass horizontal positioned, drip Rui Shi-Ji's nurse Sa A liquid (about 0.5 ~ 0.8 ml) on slide glass, and allow cover whole specimen staining, dyeing 1min;
(2) again Rui Shi-Ji's nurse Sa B solution is dripped above slide glass (amount of dropping is 2 ~ 3 times of A liquid), blowing with rubber pipette bulb makes A, B dye liquor fully mix, dyeing 3 ~ 10min;
(3) under slide glass being placed in water tap, with dye liquor 30 second that weep tap water is remaining, air-dry, Microscopic observation, gathers image.
Result is as accompanying drawing illustrates shown in middle Fig. 3, hemopoietic stem cell in embodiment 1 step (3), can be specific to erythroid differentiation, show by carrying out Rui Shi-Giemsa staining cell to the cell of cultivation the 1st day (figure A), 6 days (figure B), 10 days (figure C), 12 days (figure D), it is very large that cultured cells karyon is carried out in firm separation, occupy cell population long-pending more than 90%, kernel is obvious, each nucleus about has 3 ~ 5, the very little dye of cytoplasmic ratios is blue, presents the feature (figure A) of typical hematopoietic stem/progenitor.Along with the prolongation of induction time, cytoplasm proportion increases gradually, but nucleus still accounts for sizable ratio (figure B), when inducing culture was to 10th ~ 12 days (figure C, D), cell presents the feature of typical pronormoblast: karyon is circular, is partial between two parties or slightly on one side, account for 4/5 of cell, red-purple, chromatin particulate state, kernel is obvious.Kernel 1-2, size is uneven.Endochylema is blue, without particle, can have pseudo-Microfilament, visible understain district of nearly nucleus place.Illustrate, this induction system can break up to CFU-E by specific inducing hematopoietic stem cell.
3, the semi-solid Clone forming Test of the cell of inducing culture
Be inoculated in semisolid medium by the CFU-E being induced to the 7th day in embodiment 1 step (4), 20ml semisolid medium moiety refers to as follows: 0.9% methylcellulose gum, 25%IMDM, 5%PFHM-II, 15% foetal calf serum, 1% BSA, 1mM are dual anti-, 2 mM L-glutaminate, 200 μ g/ml Transferrins,iron complexess, 2mM Sodium.alpha.-ketopropionate, MTG(stoste 1:100 dilute).Through the cultivation of 14 days, result as shown in Figure 4, occur a large amount of red erythrocytic burst-forming units (BFU-E) and erythrocytic colony forming unit (CFU-E) (in accompanying drawing explanation Fig. 4 A, 4B) in visible semisolid medium, the ratio accounting for clone's sum through counting BFU-E and CFU-E sum is greater than 97%.Illustrate that the induction overwhelming majority through 7 days is divided into erythroid cells.We dye to the cell of redness clone, and result, as accompanying drawing illustrates shown in middle Fig. 5, has a large amount of polychromatophilic erythroblasts (in figure shown in hollow black arrow) and acidophilic normoblast (in figure, filled black arrows is shown) as seen in it.From cell dyeing result, the red system induction system that we set up can specific inducing hematopoietic stem cell to erythroid differentiation.
4, the change of Flow cytometry different time CFU-E surface marker
Collect the cell of the 5th, 7,10,14 respectively, the hematopoiesis of its surface expression and red system surface marker are detected.Result shows, through the induction of 5 days, all cells did not all express CD34, illustrated that in this induction system, original hemopoietic stem cell breaks up, and the expression of CD117 also maintains lower level, also demonstrate culturing process cell differentiation and maturation gradually from another aspect.
The expression of the specificity marker CD36 of CFU-E increases gradually along with the prolongation of induction time, and the 5th day CD36+ cell proportion is increase to 80% on the 55.8%, 7th day, increases to more than 90% after the 10th day; Show the prolongation along with incubation time, cell increases to erythroid differentiation ratio.Late period erythrocytic specific surfaces mark glycophorin A (Glycophorin A, Gly-A) expression also increases gradually with the prolongation of incubation time, by the 5th day 21.4% increase to the 7th day 42.4%, increase gradually, increased to about 75% to the 10th day, illustrate that this culture system promotes that erythroid cells breaks up to maturation; The expression of TfR CD71 also experienced by similar expression process, by the 5th day 55% increase to the 7th day close to 90%, be greater than the expression of 90% after the 10th day.Show induction system inner cell can specificity ripe to erythroid differentiation.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. an external evoked ES cell differentiation is the method for erythroid cells, it is characterized in that, the induction system containing dexamethasone is adopted to carry out pre-induced to embryonic stem cell, and then adopt the serum free medium system inducing culture containing serum-free culture additive N2 or B27, prostaglandin E2 and L-glutaminate, then adopt the differentiation-inducing one-tenth erythroid cells of serum free medium system containing STEM CELL FACTOR (SCF), interleukin-13, erythropoietin.
2. a kind of external evoked ES cell differentiation according to claim 1 is the method for erythroid cells, it is characterized in that, the erythroid cells surface marker CD36 positive expression rate that described method is differentiated to form is more than 90%.
3. a kind of external evoked ES cell differentiation according to claim 1 is the method for erythroid cells, it is characterized in that, the erythroid cells that described method is differentiated to form be a kind of for losing blood, ischemic, fluid loss, need the source supplementing the necessary colloidal solution of human body in time, be by human embryo stem cell in vitro directed differentiation formed, and substitute blood products by the method for venoclysis.
4. a kind of external evoked ES cell differentiation according to claim 1 is the method for erythroid cells, it is characterized in that, is made up of following step:
(1) the external cellar culture of embryonic stem cell;
(2) pre-induced of embryonic stem cell: adopt by induction system I cultivation be prepared from containing dual anti-DMEM/F-12 perfect medium that with the addition of 0.1mM dexamethasone;
(3) cell directional of pre-induced is divided into hemopoietic stem cell: adopt comprise volume ratio concentration be 2% N2 or B27, containing dual anti-serum-free StemSpan ACF substratum, and with the addition of 20ng/ml prostaglandin E2, the L-glutaminate of 2mM is prepared from induction system II cultivation;
(4) hemopoietic stem cell directed differentiation becomes erythroid cells: adopt comprise volume ratio concentration be 2% N2 or B27, containing dual anti-serum-free StemSpan ACF substratum, and with the addition of 0.1mM STEM CELL FACTOR (SCF), induction system III cultivation that 0.15mM interleukin-13,0.05mM erythropoietin are prepared from.
5. a kind of external evoked ES cell differentiation according to claim 4 is the method for erythroid cells, it is characterized in that, described step (1) specifically comprises the following steps: cellar culture embryonic stem cell carries out going down to posterity and increasing, this stage substratum is: substratum based on 85%DMEM substratum, add 15% Knockout Serum Replacement, 1mmol/L indispensable amino acid, 100IU/mL penicillin, 50 μ g/mL Streptomycin sulphates and 4ng/mL Prostatropin; Cell cultures, at 37 DEG C, is hatched under the carbon dioxide conditions of 5%, goes down to posterity weekly once.
6. a kind of external evoked ES cell differentiation according to claim 4 is the method for erythroid cells, it is characterized in that, described step (2) specifically comprises the following steps: in order to inducing human embryo stem cell is to the differentiation of hematopoietic cell, within 6 ~ 7 days, induces embryonic stem cell in cultivation; At 37 DEG C, digest 3 ~ 5min with 1mg/mL type Ⅳ collagenase and blow and beat into small cell cluster, then ultralow 6 well culture plates that stick are used to suspend 7 days, within 1st ~ 5 days, adopt containing 100IU/mL penicillin, the DMEM/F-12 complete culture solution of 50 μ g/mL Streptomycin sulphates is cultivated, within every 2 days, change liquid once, adopted at the 5th day that cultivates the induction system I adding pre-induced factor 0.1mM dexamethasone to cultivate 2 ~ 3 days, be placed in 37 DEG C, cultivate in 5% CO2gas incubator.
7. a kind of external evoked ES cell differentiation according to claim 4 is the method for erythroid cells, it is characterized in that, described step (3) specifically comprises the following steps: the 96 hole tissue culturing plates described step (2) pre-induced cultured cells being moved to 0.1% gelatin bag quilt; Adopt induction system II, the allos may brought because adding serum can be avoided to pollute, and put 37 DEG C, cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 2 days once; Within every 7 days, change liquid once, after adherent culture, every day observes cell growth status in culture dish under an optical microscope, cultivates and obtains the hemopoietic stem cell become by Oriented Differentiation of Embryonic Stem Cell afterwards in 20 days.
8. a kind of external evoked ES cell differentiation according to claim 4 is the method for erythroid cells, it is characterized in that, described step (4) specifically comprises the following steps:
The first step, is undertaken stem cell prepared by described step (3) through flow cytometer detection, and CD34 positive cell ratio is more than 90%, meets next step carries out amplification cultivation requirement to it;
Second step, successfully isolate CD34 positive cell by immunomagnetic beads method to be inoculated in culture dish and to cultivate, add 0.25% trypsinase-EDTA Digestive system 1.0ml, immerse and cover at the bottom of bottle, observe when seeing the rounded levitating of cell under inverted microscope, add 1.0ml foetal calf serum and stop digestion;
3rd step, adds 10ml PBS and repeatedly blows and beats flushing, at room temperature 900 revs/min centrifugal 10 minutes; After abandoning supernatant, add 10ml serum-free induction system III re-suspended cell, renewed vaccination is in culture dish;
4th step, puts 37 DEG C by above-mentioned culture dish, and cultivate 6 ~ 8 days in 5% CO2gas incubator, fluid infusion in every 3 days once; Within every 7 days, change liquid once, cultivate the erythroid cells obtaining afterwards for 20 days and come by the thin directed differentiation of Hematopoietic Stem, collect erythroid cells and carry out flow cytometer detection, result display CD36 positive expression rate is more than 90%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510131696.6A CN104694471A (en) | 2015-03-25 | 2015-03-25 | Method for inducing embryonic stem cells to be differentiated into erythroid cells in vitro |
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| CN106834224A (en) * | 2016-11-18 | 2017-06-13 | 西北农林科技大学 | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell |
| CN106854639A (en) * | 2015-12-08 | 2017-06-16 | 中国药科大学 | One kind is based on three-dimensional semisolid adult stem cell cultural method |
| CN107254442A (en) * | 2017-08-08 | 2017-10-17 | 安徽惠恩生物科技股份有限公司 | A kind of artificial induction's pluripotent stem cell differentiation is the method for neural precursor |
| CN107287159A (en) * | 2017-08-23 | 2017-10-24 | 安徽惠恩生物科技股份有限公司 | A kind of artificial induction's pluripotent stem cell differentiation is the method for candidate stem cell |
| CN109157690A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | Monkey-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain |
| CN109157693A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | The preparation of people-mouse cell fusion mother's tire blood group incompatibility treatment hybrid strain |
| CN109157692A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | People-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain |
| CN109157691A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | The preparation of monkey-mouse cell fusion mother's tire blood group incompatibility treatment hybrid strain |
| CN110923195A (en) * | 2019-12-31 | 2020-03-27 | 南昌诺汇医药科技有限公司 | Efficient induction differentiation culture method of embryonic stem cells |
| CN112143697A (en) * | 2020-10-08 | 2020-12-29 | 北京广未生物科技有限公司 | Method for promoting proliferation and differentiation of embryonic stem cells |
| CN113373114A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN113373115A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and culture method for differentiating hematopoietic stem cells by using low-cost pluripotent stem cells |
| CN114438036A (en) * | 2021-12-03 | 2022-05-06 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting directional differentiation and maturation of stem cells into erythroid cells and application |
| WO2024108461A1 (en) * | 2022-11-24 | 2024-05-30 | 苏州血霁生物科技有限公司 | Method for platelet differentiation, culture medium, and use thereof |
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| CN106854639A (en) * | 2015-12-08 | 2017-06-16 | 中国药科大学 | One kind is based on three-dimensional semisolid adult stem cell cultural method |
| CN106834224A (en) * | 2016-11-18 | 2017-06-13 | 西北农林科技大学 | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell |
| CN107254442A (en) * | 2017-08-08 | 2017-10-17 | 安徽惠恩生物科技股份有限公司 | A kind of artificial induction's pluripotent stem cell differentiation is the method for neural precursor |
| CN107287159A (en) * | 2017-08-23 | 2017-10-24 | 安徽惠恩生物科技股份有限公司 | A kind of artificial induction's pluripotent stem cell differentiation is the method for candidate stem cell |
| CN109157690A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | Monkey-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain |
| CN109157693A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | The preparation of people-mouse cell fusion mother's tire blood group incompatibility treatment hybrid strain |
| CN109157692A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | People-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain |
| CN109157691A (en) * | 2018-06-15 | 2019-01-08 | 翁炳焕 | The preparation of monkey-mouse cell fusion mother's tire blood group incompatibility treatment hybrid strain |
| CN110923195A (en) * | 2019-12-31 | 2020-03-27 | 南昌诺汇医药科技有限公司 | Efficient induction differentiation culture method of embryonic stem cells |
| CN110923195B (en) * | 2019-12-31 | 2020-11-17 | 广东赛尔生物科技有限公司 | Efficient induction differentiation culture method of embryonic stem cells |
| CN112143697A (en) * | 2020-10-08 | 2020-12-29 | 北京广未生物科技有限公司 | Method for promoting proliferation and differentiation of embryonic stem cells |
| CN113373114A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells |
| CN113373115A (en) * | 2021-06-11 | 2021-09-10 | 杭州原生生物科技有限公司 | Culture medium and culture method for differentiating hematopoietic stem cells by using low-cost pluripotent stem cells |
| CN114438036A (en) * | 2021-12-03 | 2022-05-06 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting directional differentiation and maturation of stem cells into erythroid cells and application |
| CN114438036B (en) * | 2021-12-03 | 2023-08-18 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting directional differentiation and maturation of stem cells into erythroid cells and application |
| WO2024108461A1 (en) * | 2022-11-24 | 2024-05-30 | 苏州血霁生物科技有限公司 | Method for platelet differentiation, culture medium, and use thereof |
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