CN102321571A - Method for preparing autologous hematopoietic stem cell by differentiated cell - Google Patents
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Abstract
The invention discloses a method for preparing autologous hematopoietic stem cells by differentiated cells, which comprises the following steps: a. obtaining hematopoietic stem cells from a placenta; b. denucleating the hematopoietic stem cells to obtain cytoplasm; c. fusing the obtained cytoplasm and differentiated cells to generate cybrid cells; d. culturing the cybrid cells to generate hematopoietic stem cells. The invention uses the principle of cytoplasmic interaction, allows the separated and denucleated hematopoietic stem cells to interact with fibroblast nucleuses, reprograms the nucleuses, generates fused hematopoietic stem cells, can construct fused heterozygous hematopoietic stem cells which is consistent with patient HLA matched-type, and the constructed hematopoietic stem cells can be used as a new source of clinical hematopoietic stem cell transplantation. The method of the invention is simple, economical, and convenient, provides new basic information and theoretical basis for the treatment of diseases such as leukemia and the like, and provides a new way for the generation of patient autologous hematopoietic stem cells in clinic.
Description
Technical field
The present invention relates to a kind ofly prepare the method for autologous stem cell, belong to the gene engineering field from noble cells.
Background technology
Stem cell is the not specific cell of a kind of function; Have two characteristics: promptly have unlimited self ability; Can be divided into the ability of more than one height differone cell, in fact comprised from fetal development to adult growth and development process various not differentiation and maturation cells.Both comprised the origin of life cell, the initiating cell that histoorgan is grown comprises that also adult tissue updates, the seed cell of injury repairing.Therefore, stem cell has multiple potential purposes, comprises cell therapy, is used to make tissue and organ, gene therapy and the drug screening etc. that can supply transplant.
Adult stem cell is specific differentiation or the undifferentiated cell of one type of function, and it is present in the multiple adult tissue.Adult stem cell can break up the nearly all cell type that becomes in the affiliated tissue, and can stride the germinal layer differentiation, has multidirectional differentiation capability.Adult stem cell is common in marrow, blood, brain, cornea, tooth, liver, skin etc.
Have a large amount of hemopoietic stem cells in the placenta, can be used for transplanting treating hematologic disease, have very important clinical application to the patient who suffers from diseases in the blood system such as serious white blood disease.But have two shortcomings, the one, cell concentration is less relatively, children's consumption can, then measure wretched insufficiency but the adult used, can not be used for the adult and transplant; The second, the most important thing is that the placenta hemopoietic stem cell has the HLA (HLA) of oneself, though immunogenicity is less, also there is the immunosuppression reaction.Since hemopoietic stem cell has important purposes, can make up the patient's of the HLA with oneself hemopoietic stem cell, solve immunosuppression danger? Therefore the demand that has the preparation autologous stem cell.
Summary of the invention
The present invention is based on such understanding; It is the cell generation reprogrammed that the kytoplasm of existing embryonic stem cell can make differentiation; Become multipotential stem cell, whether does the kytoplasm of multipotential stem cell also have such function so? And, if reprogrammed takes place; Whether the hybrid cell that obtains has identical HLA with the donor's cells; Promptly have immune compatibility, can the new multipotential stem cell that produces be differentiated to form and the identical HLA in confession cell source, can avoid the immunological rejection that in patient's body, produces thus.Therefore, the present invention is intended to solve solution immunological rejection above-mentioned and source of human stem cell problem, and a kind of method for preparing autologous stem cell from noble cells is provided.
Technical scheme of the present invention is: a kind ofly prepare the method for autologous stem cell from noble cells, may further comprise the steps:
A, from placenta, obtain hemopoietic stem cell;
B, with the hemopoietic stem cell stoning, obtain kytoplasm;
C, obtaining kytoplasm and noble cells are merged, to produce cybrid;
D, the said cybrid of cultivation are to produce hemopoietic stem cell.
In addition, before the d step, can also add that said cybrid is transferred to the step that increases on the feeder layer, described feeder layer can be placenta attached cell feeder layer.
Above-mentioned noble cells comprises differentiation cells such as SF, hair follicle cell, hair follicle stem cells.
The present invention utilizes kytoplasm to make principle mutually, with the umbilical hemopoietic stem cell separation and purification, and the density gradient centrifugation stoning; The hemopoietic stem cell kytoplasm can with the inoblast nuclear interaction, the pair cell nuclear reprogramming produce to merge hemopoietic stem cell; Can make up with patient HLA and join the consistent fusion heterozygosis hemopoietic stem cell of type,, can be used as a new source of clinical transplantation hemopoietic stem cell through the screening amplification; Its wide material sources, and do not have immunological rejection.The inventive method is simple, and economical convenient, for treatment of diseases such as white blood disease provide new basic data and theoretical foundation, more clinical generation patient autologous stem cell provides a new mode.
Below in conjunction with accompanying drawing and embodiment the present invention is at length explained; Though the present invention allows multiple multi-form embodiment; But the present invention will specify at least a embodiment and other possible alternative methods; Simultaneously, content disclosed by the invention should be considered to be in and illustrate method of the present invention and principle, rather than limits the present invention in the described embodiment.
Description of drawings
Fig. 1 merges the back hemopoietic stem cell CD 34
+Each stage morphological feature that cells in vitro is cultivated.
Embodiment
The main thought that the present invention produces autologous stem cell is: will merge and the generation cybrid from the hemopoietic stem cell and the noble cells of placenta; It is a multipotential stem cell; The cybrid that is produced is expressed the major histocompatibility antigen (MHC) in donorcells source, particularly expresses the HLA (HLA) identical with said cell.
The noble cells that the present invention uses can be SF, hair follicle cell, hair follicle stem cells etc., and does not use lymphocyte.This is that nocuity is less because relative other cells sources of SF are fairly simple.Therefore and kytoplasm is less, and it is proper using the kytoplasm of SF and hemopoietic stem cell to merge.
One, from Cord blood and placenta, obtains hemopoietic stem cell
1, the collection of Cord blood (UCB)
Syringe with 60ml sucks the 2ml heparin, loads onto the syringe needle of 16 bores, from umbilical vein, collects UCB, puts upside down immediately and carries out mixing several times, prevents blood coagulation.
2, the preparation of MNC and cultivation
(1) in each 50ml centrifuge tube, adds 15mlUCB, dilute with 20mlPBS.
(2) add 15mlFicoll, with the bottom that Ficoll places UCB, make it to form an interface clearly with conduit.
(3) centrifugal 740g, room temperature 30min.
(4) after centrifugal, MNC forms the flaxen vaporific suspended layer of one deck between the FIcoll of the yellow serum layer on top and bottom, be placed on the flaxen suspended layer with suction nozzle, and sucking-off MNC changes MNC in the centrifuge tube that substratum is housed over to then gently.
(5) with MNC in room temperature with the centrifugal 10min of 515g, supernatant discarded.Sedimentary cell mass is dispelled gently re-suspended cell.
(6) repeating step 5 once.
(7) with MNC suspension mixing, get the 30ul cell, the blue dyeing of placenta, counting.
3, magnetic sorting hemopoietic stem cell (CD34+ cell)
(1) using PBS adjustment MNCs concentration is 2 * 10
8/ ml, the transitional cell suspension is to the Vestolen PP 7052 Falcon pipe of 5ml;
(2) add EasySep Human CD34 Positive Selection Cocktail 10 μ l, act on 15min under the room temperature behind the mixing gently;
(3) add EasySep Magnetic Nanoparticles 5 μ l, act on 10min under the room temperature behind the mixing gently; Add PBS and complement to 2.5ml, mixing;
(4) the Falcon pipe is placed EasySep Magnet magnetic field, room temperature leaves standstill 5min, and liquid inclines; PBS repetitive scrubbing 4 times, an amount of PBS mixing cell, counting.
Two, with the hemopoietic stem cell stoning, obtain kytoplasm
1, hemopoietic stem cell (CD34+ cell) is earlier hatched 5min in no calcium magnesium PBS () solution after, warp 0.25% trypsinase+0.04%EDTA solution is digested to individual cells again.
2, add among the DMEM and digestion; The centrifugal 5min of 1500r/min discards supernatant, in centrifuge tube, adds the DMEM nutrient solution that 1ml contains cytochalasin B and 0.5%DMSO again; Again process cell suspension; Auto-regulating System of Density of Heavy Medium to 1 * 10/ml are put incubator and are cultivated 1h, after move to off-the-shelf gradient column top again.
It is centrifugal that the Ficoll density gradient centrifugation pipe that 3, will contain the CD34+ cell suspension is put the supercentrifuge that is incubated to 30 ℃ in advance, and rotating speed is 25000r/min, centrifugal 50min.
4, after the centrifugal end,, add 20ml DMEM nutrient solution again with the whole impouring small beakers of the solution in the centrifuge tube, centrifugal 1500r/min, 5min abandons and adds 1ml DMEM nutrient solution after the supernatant, and this cell fragment suspension is moved into four orifice plates, puts incubator and cultivates.
Three, obtaining cell cytoplasm and noble cells are merged, produce cybrid
1, noble cells (inoblast) obtains
(1) operation cuts the skin 2-3cm of patient's belly or lower limb
2, place the PBS that contains 100IU/ml penbritin and 100mg/ml Vetstrep, after the PBS rinsing several times, cut and abandon fat and reticular tissue repeatedly.Skin is cut into 1mm
3About the fritter of size, add and to contain among the DMEM of collagenase, Unidasa, place 4 degree refrigerator overnight.
(2) add the Digestive system that contains 0.25% pancreatin and 0.02%EDTA, 37 ℃ of digestion 20min add and contain the termination of serum nutrient solution.The centrifugal 10min collecting cell of 1200rpm.DMEM with containing 15% foetal calf serum cultivates inoculating cell density 2 * 105, cell inoculation, 37 ℃, 5%CO
2, CO
2Cultivate under the incubator condition.
(3) cultivation of going down to posterity of SF, after the cultured skin inoblast grew up to individual layer, the PBS rinsing of the no calcium ions and magnesium ions of usefulness added the Digestive system digestion that contains 0.25% pancreatin and 0.02%EDTA, 2-5min several times.Add the DMEM substratum piping and druming contain foetal calf serum and stop digestion, go down to posterity, place 37 ℃, 5%CO with the ratio of 1 ︰ 3
2, CO
2Cultivate under the incubator condition.
2, kytoplasm and noble cells merge
(1) kytoplasm of taking-up separator well, with cultured inoblast, the DMEM with serum-free washes twice again, centrifugal respectively and counting.At ambient temperature, in centrifuge tube, mix two kinds of cells, centrifugal then 10min, 1000 commentaries on classics/min by 10 ︰ 1.
(2) remove supernatant, the button centrifuge tube that shakes scatters the cell sediment gently, splashes into fusogen (50% polyoxyethylene glycol PEG solution) with suction pipe, 0.5ml, and rotating centrifugal pipe or stir gently with pipette tip increases the chance of cellular exposure, contact PEG as far as possible.
(3) drip (fusion process is no more than 6 ~ 7 min) behind 10 serum-free mediums continuously, the centrifugal supernatant that goes adds the DMEM nutrient solution that 10ml contains 10%FCS again and suspends centrifugal 10min.
(4) remove the cell sediment of supernatant, wash twice with the DMEM nutrient solution that contains 10%FCS after, resuspending.And at 37 ℃, 5%CO
2, saturated humidity CO
2Cultivate in the incubator.Inverted microscope is observation of cell form and growth profile day by day.And record is taken pictures.
Four, cultivate institute's above-mentioned impurity cell that obtains to produce hemopoietic stem cell.
Before above-mentioned (four) step, can also adopt following steps that said cybrid is transferred on the feeder layer and increase, feeder layer can be placenta attached cell feeder layer.
One, the making of placenta attached cell feeder layer
1, aseptic condition separates healthy term birth of newborn fresh fetal placental villi film leaflet tissue down, uses D-Hank ' s liquid to wash repeatedly, and the chorion tissue is shredded, and 4 ℃ of digestion of pancreas enzyme-EDTA are spent the night;
2, tissue block is placed contain 15% serum, 2mM/L Stimulina, 100 U/ml penicillium mould, in the α of 100 μ g/ml Streptomycin sulphates-MEM substratum, at 5% CO
2, cultivate under the saturated humidity, 37 ℃ of conditions.
3, whenever change liquid once at a distance from 3～4 d; Treating that cytogamy reaches at 70～80% o'clock, goes down to posterity by 1 ︰ 2 in conventional digestion back, and the petridish bottom is placed slide and encapsulated with gelatin; Get the cell that goes down to posterity more than 3 times and carry out immunodetection and induce differentiation to identify, MTC is handled for use.
Two, the amplification of impurity cell on placenta attached cell feeder layer
The impurity cell is placed on the above-mentioned feeder layer, be inoculated in the culturing bottle.Nutrient solution includes 10ml IMDM, at 37 ℃, and 5%CO
2, cultivate amplification under the saturated humidity.
Experimental result is with reference to shown in Figure 1.
Fig. 1 merges the back hemopoietic stem cell CD 34
+Each stage morphological feature that cells in vitro is cultivated, wherein:
A: hemopoietic stem cell CD 34
+Cells in vitro is cultivated 2d, visible cell division (400 *);
B: hemopoietic stem cell CD 34
+Cells in vitro is cultivated 3d, visible cell bunch formation (400 *);
C, D: hemopoietic stem cell CD 34
+Cells in vitro is cultivated 6d, and visible fine and close cell colony forms (200 *);
E, F: hemopoietic stem cell CD 34
+Cells in vitro is cultivated 10d, and visible CFU-GEMM forms (100 *).
Claims (4)
1. one kind prepares the method for autologous stem cell from noble cells, may further comprise the steps:
A, from placenta, obtain hemopoietic stem cell;
B, with the hemopoietic stem cell stoning, obtain kytoplasm;
C, obtaining kytoplasm and noble cells are merged, to produce cybrid;
D, the said cybrid of cultivation are to produce hemopoietic stem cell.
2. according to claim 1ly prepare the method for autologous stem cell, it is characterized in that, said cybrid is transferred to the step that increases on the feeder layer before being included in the d step from noble cells.
3. according to claim 2ly prepare the method for autologous stem cell, it is characterized in that said feeder layer is a placenta attached cell feeder layer from noble cells.
4. according to claim 1ly prepare the method for autologous stem cell, it is characterized in that said noble cells comprises SF, hair follicle cell, hair follicle stem cells from noble cells.
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| CN201110281117A CN102321571A (en) | 2011-09-21 | 2011-09-21 | Method for preparing autologous hematopoietic stem cell by differentiated cell |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967683A (en) * | 2017-05-09 | 2017-07-21 | 林涛 | The method for cultivating pluripotential hemopoietic stem cell |
| CN109735492A (en) * | 2019-03-06 | 2019-05-10 | 福建省海西细胞生物工程有限公司 | A kind of candidate stem cell extracting method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362965A (en) * | 1999-06-30 | 2002-08-07 | 先进细胞技术公司 | cytoplasmic transfer to de-differentiate recipient cells |
| CN1997275A (en) * | 2004-05-13 | 2007-07-11 | 生殖遗传学研究所 | Method of making stem cells from differentiated cells |
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| CN1362965A (en) * | 1999-06-30 | 2002-08-07 | 先进细胞技术公司 | cytoplasmic transfer to de-differentiate recipient cells |
| CN1997275A (en) * | 2004-05-13 | 2007-07-11 | 生殖遗传学研究所 | Method of making stem cells from differentiated cells |
| WO2008103462A2 (en) * | 2007-02-23 | 2008-08-28 | Advanced Cell Technology, Inc. | Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106967683A (en) * | 2017-05-09 | 2017-07-21 | 林涛 | The method for cultivating pluripotential hemopoietic stem cell |
| CN109735492A (en) * | 2019-03-06 | 2019-05-10 | 福建省海西细胞生物工程有限公司 | A kind of candidate stem cell extracting method |
| CN109735492B (en) * | 2019-03-06 | 2022-10-04 | 福建省海西细胞生物工程有限公司 | Hematopoietic stem cell extraction method |
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