CN108676777A - Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation - Google Patents
Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation Download PDFInfo
- Publication number
- CN108676777A CN108676777A CN201810288645.8A CN201810288645A CN108676777A CN 108676777 A CN108676777 A CN 108676777A CN 201810288645 A CN201810288645 A CN 201810288645A CN 108676777 A CN108676777 A CN 108676777A
- Authority
- CN
- China
- Prior art keywords
- cell
- tire
- stem cell
- sinusoidal endothelial
- optionally
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention proposes purposes of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment.Tire sinusoidal endothelial cell can be that candidate stem cell builds microenvironment, the amplification in vitro of hematopoiesis support stem cell, and promotes pluripotent stem cell to break up to HSCs or directly reprogram body cell and obtain HSCs.
Description
Technical field
The present invention relates to biological fields.In particular it relates to which tire sinusoidal endothelial cell strain is in hematopoietic stem cell expansion
In application.
Background technology
Candidate stem cell (hematopoietic stem cell, HSC) has the self-renewal capacity of height and multidirectional
Differentiation potential can generate all types of blood cells, such as red blood cell, leucocyte, blood platelet and lymphocyte.It is in life
Entire hemopoietic system can not only be rebuild during life, be also equipped with the function of maintaining long term hematopoietic.In recent years, HSC transplanting is more next
It is applied to the malignant tumour of clinical treatment blood or non-blood system more, shows wide application prospect.Wherein, especially
Favor is received with Umbilical Cord Blood Transplantation (umbilical cord blood transplantation, UCBT), it has source wide
It is general, be easy to acquisition, low, relapse after transplantation rate and graft versus host disease(GVH disease) (graft- required to donor fanout free region, HLA distribution type
Versus-host disease, GVHD) incidence it is relatively low the advantages that.However, HSC absolute quantities are less in single cord blood,
The risk that neutrophil leucocyte restores to postpone and increase bacterium and virus infection is easy to cause after infusion, and double Umbilical Cord Blood Transplantation is then
A series of problems, such as increase of graft versus host disease(GVH disease) incidence, platelet recovery time lengthening can be brought.In general, to HSCs
It is most direct easily solution to carry out amplification in vitro culture, but builds suitable HSC amplification in vitro microenvironments so far
It is still bottleneck problem urgently to be resolved hurrily.
Invention content
The present invention is directed to solve at least one the technical problems existing in the prior art at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes：
Schofield has been put forward for the first time the concept of stem cell niche (Niche) within 1978, it is indicated that specific microenvironment
(Microenvironment) stem cell function can be acted, then there is lot of documents to confirm stem cell in Various Tissues
The presence of tabernacle.People once attempted various ways and carry out hematopoieticmicroenviron-ment in analogue body, with it is expected build maintain HSCs self-renewings with
The culture system in vitro of amplification, such as the relevant cell factor of hematopoiesis, small molecule, stroma cell co-culture, Notch ligands, but
Effect is still barely satisfactory.
Hematopoieticmicroenviron-ment is made of a variety of sertoli cells for closing on HSCs, including osteoblast, endothelial cell, fat are thin
Born of the same parents, stroma cell and immunocyte etc., wherein endothelial cell possess common-ancestor cell-hemogenic endothelium, many tables of coexpression with HSCs
Face mark and transcription factor such as CD34, CD31, Runx1, GATA-2 etc., and can pass through and secrete a variety of Effects of Factors HSCs'
Proliferation and self-renewing, become the core component of hematopoieticmicroenviron-ment.
In embryonic development, liver is that an important blood forming organ and HSCs self-renewings and amplification are most vigorous
Place, tire sinusoidal endothelial cell therein (Human fetal liver sinusoid endothelial cells,
HFLSECs) be a kind of unique structure blood sinus endothelium, account for about the 70% of liver non-parenchymal cell sum, have that phagocytosis, antigen carries
It is in, the functions such as blood flow regulation, ET-1, NO, vascular endothelial growth factor (VEGF), transforminggrowthfactor-β1 (TGF-β can be secreted
1), the cytokine profiles such as tumor necrosis factor (TNF) also participate in extramedullary hematopoiesis and HSCs and are planted in the selectivity in lobuli hepatis area
Enter.
In view of this, inventor uses tire sinusoidal endothelial cell as feeder cells, to build candidate stem cell micro-loop
Border promotes hematopoietic stem cell expansion.Further, inventor has found, since the survival of tire sinusoidal endothelial cell depends on serum
And endothelial growth factor, but the amplification environmental requirement serum-free of candidate stem cell and without endothelial growth factor, from
And it will appear tire sinusoidal endothelial cell apoptosis after causing tire sinusoidal endothelial cell to co-culture the short period with candidate stem cell.
E4orf1 is the gene outcome of the code areas adenovirus E4, regulating cell signal path and then can influence the cell cycle
And Apoptosis.Inventor has found, by E4orf1 channel genes tire sinusoidal endothelial cells, tire sinusoidal endothelial cell is enabled to exist
Under conditions of serum-free and endothelial growth factor survive and maintain self-characteristic, and need not move through mitomycin C or
The pretreatment of emitting radiation can maintain the existing state not being proliferated.So that microenvironment is built for candidate stem cell, conducive to making
The amplification of hemocytoblast, and promote pluripotent stem cell to break up to HSCs or directly reprogram body cell and obtain HSCs.
For this purpose, in one aspect of the invention, it is micro- in structure candidate stem cell that the present invention proposes tire sinusoidal endothelial cell
Purposes in environment.Tire sinusoidal endothelial cell have the function of phagocytosis, antigen offer, blood flow regulation etc., can secrete ET-1, NO,
The various kinds of cell such as vascular endothelial growth factor (VEGF), transforminggrowthfactor-β1 (TGF-β 1), tumor necrosis factor (TNF) because
Son also participates in extramedullary hematopoiesis and HSCs being selectively implanting in lobuli hepatis area.In turn, inventor has found, tire hepatic sinusoidal endothelium is thin
Born of the same parents can be candidate stem cell build microenvironment, the amplification in vitro of hematopoiesis support stem cell, and promote pluripotent stem cell to
HSCs breaks up or directly reprogramming body cell obtains HSCs.
According to an embodiment of the invention, the purposes can also have following additional technical feature：
According to an embodiment of the invention, the tire sinusoidal endothelial cell is provided in the form of recombinant cell, described heavy
Group cell carries E4orf1 genes and optional GFP genes.
According to an embodiment of the invention, the tire sinusoidal endothelial cell is used as feeder cells.
According to an embodiment of the invention, the tire sinusoidal endothelial cell is for maintaining hematopoietic stem cell expansion.
According to an embodiment of the invention, on the recombinant cell endothelial progenitor cells mark CD133, dryness mark CD117 with
And the expression rate of hematopoietic cell mark CD45 is below 0.4%, vascular endothelial growth factor receptor KDR expression rates are 55~
80%, mark CD144 and the CD31 expression rate of endothelial cell is all higher than 99%.
In still another aspect of the invention, the present invention proposes a kind of recombinant cell.According to an embodiment of the invention, described heavy
Group cell is the tire sinusoidal endothelial cell for carrying E4orf1 genes.Inventor has found, due to the survival of tire sinusoidal endothelial cell
Dependent on serum and endothelial growth factor, but the amplification environmental requirement serum-free of candidate stem cell and given birth to without endothelial cell
It is thin to will appear tire hepatic sinusoidal endothelium so as to cause tire sinusoidal endothelial cell and after the candidate stem cell co-cultivation short period for the long factor
Born of the same parents' apoptosis.Further, inventor has found through further investigation, by E4orf1 channel genes tire sinusoidal endothelial cells so that tire liver
Sinusoidal endothelial cells can survive under conditions of serum-free and endothelial growth factor and maintain self-characteristic, and without warp
The existing state not being proliferated can be maintained by crossing the pretreatment of mitomycin C or emitting radiation, to build promotion Hematopoietic Stem
The microenvironment of cell expansion ex vivo is conducive to the amplification of candidate stem cell, and promotion pluripotent stem cell to HSCs differentiation or directly
It connects reprogramming body cell and obtains HSCs.
According to an embodiment of the invention, the tire sinusoidal endothelial cell carries GFP genes.
According to an embodiment of the invention, the recombinant cell is used as feeder cells.
In still another aspect of the invention, the present invention proposes a kind of method preparing recombinant cell noted earlier.According to this
The embodiment of invention, the method includes：The first vector for carrying E4orf1 genes is added and contains the tire hepatic sinusoidal endothelium
It in first culture medium of cell, is cultivated, to obtain the recombinant cell.Recombination according to the ... of the embodiment of the present invention as a result,
Cell can be candidate stem cell build microenvironment, be conducive to candidate stem cell amplification, and promote pluripotent stem cell to
HSCs breaks up or directly reprogramming body cell obtains HSCs.
According to an embodiment of the invention, the method further includes：The first vector and GFP bases will be carried respectively
The Second support of cause is added jointly in the culture medium of the tire sinusoidal endothelial cell.
According to an embodiment of the invention, the first vector is to be packaged with the retrovirus of plasmid MSCV-N E4orf1,
And anti-puromycin gene is carried on the plasmid.
According to an embodiment of the invention, the Second support is the retrovirus for being packaged with plasmid pMX-GFP.
According to an embodiment of the invention, the first vector and the volume ratio of Second support are 1:1.
According to an embodiment of the invention, the puromycin containing 0.5 μ g/ml in first culture medium does not contain serum
And endothelial growth factor.
According to an embodiment of the invention, first culture medium is EGM-2 basal mediums.
In still another aspect of the invention, the present invention proposes a kind of method of hematopoietic stem cell expansion.It is according to the present invention
Embodiment, the method includes：The method of Prepare restructuring cell obtains recombinant cell as described above；And it is Hematopoietic Stem is thin
Born of the same parents are inoculated in the second culture medium containing the recombinant cell, are cultivated, to make the hematopoietic stem cell expansion.
According to an embodiment of the invention, the candidate stem cell is CD34+Cell.
According to an embodiment of the invention, serum and endothelial growth factor are not contained in second culture medium, it is described
Second culture medium is StemSpan culture mediums, contains 50ng/ml SCF, 50ng/ml Flt-3L and 50ng/ml TPO.
In still another aspect of the invention, the present invention proposes a kind of kit.According to an embodiment of the invention, the reagent
Box includes：Tire sinusoidal endothelial cell or recombinant cell noted earlier.Kit energy according to the ... of the embodiment of the present invention is utilized as a result,
Enough build candidate stem cell microenvironment, be conducive to candidate stem cell amplification, and promote pluripotent stem cell to HSCs break up or
Directly reprogramming body cell obtains HSCs.
According to an embodiment of the invention, the kit is for building in the microenvironment of candidate stem cell.
According to an embodiment of the invention, the kit is for expanding the candidate stem cell.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obviously, or practice through the invention is recognized.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination following accompanying drawings to embodiment
Obviously and it is readily appreciated that, wherein：
Fig. 1 shows expression signals of the identified by immunofluorescence vWF according to an embodiment of the invention in HFLSECs
Figure；
Fig. 2 shows according to an embodiment of the invention under the condition of culture containing 0.5 μ g/ml puromycin
HFLSECs light microscopic figures, wherein left figure is puromycin pressurizations screening 2 days, and right figure is puromycin pressurizations screening 3 days；
Fig. 3 shows the light microscopic figure of the retrovirus according to an embodiment of the invention for being packaged with plasmid pMX-GFP；
Fig. 4 shows according to an embodiment of the invention under serum-free and endothelial growth factor condition of culture
The flow cytometry schematic diagram and light microscopic figure of E4orf1-GFP/HFLSECs；
Fig. 5 shows that the surface marker albumen streaming of E4orf1-GFP/HFLSECs according to an embodiment of the invention is thin
Born of the same parents analyze schematic diagram；
Fig. 6 shows E4orf1-GFP/HFLSECs according to an embodiment of the invention as feeder layer backer's umbilical cord
The CD34 in blood source+The amplification in vitro of cell analyzes schematic diagram；
Fig. 7 shows the CD34+ cells in human cord blood source according to an embodiment of the invention after amplification in vitro
Hematopoietic colonies Forming ability schematic diagram.
Specific implementation mode
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
The present invention proposes purposes, recombinant cell, system of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment
Method, the method for hematopoietic stem cell expansion and the kit of standby recombinant cell.It will be described in greater detail respectively below.
Purposes of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment
In one aspect of the invention, the present invention proposes tire sinusoidal endothelial cell in building candidate stem cell microenvironment
Purposes.Tire sinusoidal endothelial cell have the function of phagocytosis, antigen offer, blood flow regulation etc., ET-1, NO, intravascular can be secreted
The cytokine profiles such as skin growth factor (VEGF), transforminggrowthfactor-β1 (TGF-β 1), tumor necrosis factor (TNF), also join
Being selectively implanting in lobuli hepatis area with extramedullary hematopoiesis and HSCs.In turn, inventor has found, tire sinusoidal endothelial cell can be
Candidate stem cell builds microenvironment, the amplification in vitro of hematopoiesis support stem cell, and pluripotent stem cell is promoted to break up to HSCs
Or directly reprogramming body cell obtains HSCs.
According to an embodiment of the invention, tire sinusoidal endothelial cell is provided in the form of recombinant cell, and recombinant cell is taken
With E4orf1 genes and optional GFP genes.According to a particular embodiment of the invention, recombinant cell is by by E4orf1
Gene and optional GFP channel genes tire sinusoidal endothelial cells and obtain.
Inventor's discovery, since the survival of tire sinusoidal endothelial cell depends on serum and endothelial growth factor, still
The amplification environmental requirement serum-free of candidate stem cell and without endothelial growth factor, so as to cause tire sinusoidal endothelial cell with make
Hemocytoblast will appear tire sinusoidal endothelial cell apoptosis after co-culturing the short period.For this purpose, inventor leads E4orf1 genes
Enter tire sinusoidal endothelial cell so that tire sinusoidal endothelial cell can survive under conditions of serum-free and endothelial growth factor
And self-characteristic is maintained, and the pretreatment for needing not move through mitomycin C or emitting radiation can maintain what is be not proliferated to deposit
State living.To build candidate stem cell microenvironment, the amplification in vitro of hematopoiesis support stem cell, and promote pluripotency dry
Cell breaks up to HSCs or directly reprogramming body cell obtains HSCs.
In addition, in order to observe cell after conversion, and by itself and the cell differentiation for not importing target gene, inventor attempts will
Reporter gene imports tire sinusoidal endothelial cell.It finds by many experiments, is made with the GFP genes of encoding green fluorescent protein
For that in tracer channel genes tire sinusoidal endothelial cell, Detectable effects can not only be played, nor affect on the expression of E4orf1 genes.
It is particularly suitable for pluripotent stem cell and carries out area to HSCs differentiation or when directly reprogramming body cell obtains HSCs with initiator cell
Point.
According to an embodiment of the invention, it endothelial progenitor cells mark CD133, dryness mark CD117 and is made on recombinant cell
The expression rate of haemocyte mark CD45 is below 0.4%, and vascular endothelial growth factor receptor KDR expression rates are 55~80%, interior
Mark CD144 and the CD31 expression rate of chrotoplast is all higher than 99%.Recombinant cell itself is that the tire hepatic sinusoidal endothelium of primary separation is thin
Born of the same parents, inventor do it surface marker of streaming experimental verification cell, it was demonstrated that the ripe endothelium of recombinant cell height expression of the invention
The mark of cell, and endothelial progenitor cells and dryness mark are hardly expressed, the mark of hematopoietic cell is not expressed yet.As a result, with table
The recombinant cell purity of the bright present invention is higher.
According to an embodiment of the invention, tire sinusoidal endothelial cell is used as feeder cells.Inventor's discovery, will be in tire sinus hepaticus
Chrotoplast is co-cultured as feeder cells and candidate stem cell, can build candidate stem cell microenvironment, is conducive to promote its expansion
Increase and maintain self-renewing.
According to an embodiment of the invention, tire sinusoidal endothelial cell is for promoting hematopoietic stem cell expansion.Inventor's discovery, tire
Sinusoidal endothelial cell can be that candidate stem cell builds microenvironment, be conducive to promote its amplification and maintain self-renewing.
Recombinant cell
In another aspect of this invention, the present invention proposes a kind of recombinant cell.According to an embodiment of the invention, recombination is thin
Born of the same parents are the tire sinusoidal endothelial cell for carrying E4orf1 genes.Inventor has found, since the survival of tire sinusoidal endothelial cell relies on
In serum and endothelial growth factor, but the amplification environmental requirement serum-free of candidate stem cell and without endothelial cell growth because
Son is that will appear tire sinusoidal endothelial cell to wither after co-culturing the short period with candidate stem cell so as to cause tire sinusoidal endothelial cell
It dies.Further, inventor has found through further investigation, by E4orf1 channel genes tire sinusoidal endothelial cells so that in tire sinus hepaticus
Chrotoplast can survive under conditions of serum-free and endothelial growth factor and maintain self-characteristic, and need not move through silk
The pretreatment of rimocidin C or emitting radiation can maintain the existing state not being proliferated.To which structure promotes candidate stem cell
The microenvironment of amplification in vitro is conducive to the amplification of candidate stem cell, and promotion pluripotent stem cell to HSCs differentiation or directly again
It programs body cell and obtains HSCs.
According to an embodiment of the invention, tire sinusoidal endothelial cell carries GFP genes.In order to observe cell after conversion, and
By itself and the cell differentiation for not importing target gene, inventor attempts reporter gene importing tire sinusoidal endothelial cell.Through
Cross many experiments discovery, using the GFP genes of encoding green fluorescent protein as tracer channel genes tire sinusoidal endothelial cell in, no
Detectable effects can be only played, the expression of E4orf1 genes is nor affected on.It is particularly suitable for promoting pluripotent stem cell to HSCs
It is distinguished with initiator cell when differentiation or directly reprogramming body cell acquisition HSCs.
According to an embodiment of the invention, recombinant cell is used as feeder cells.Inventor has found that recombinant cell is used as raising
Confluent monolayer cells are co-cultured with candidate stem cell.Recombinant cell can survive under conditions of serum-free and endothelial growth factor
And self-characteristic is maintained, and the pretreatment for needing not move through mitomycin C or emitting radiation can maintain what is be not proliferated to deposit
State living.To build microenvironment for candidate stem cell, be conducive to the amplification of candidate stem cell, and promote pluripotent stem cell
Break up to HSCs or directly reprogramming body cell obtains HSCs.
The method of Prepare restructuring cell
In still another aspect of the invention, the present invention proposes a kind of method preparing recombinant cell noted earlier.According to this
The embodiment of invention, this method include：The first vector for carrying E4orf1 genes is added containing tire sinusoidal endothelial cell
It in first culture medium, is cultivated, to obtain recombinant cell.Inventor find, due to tire sinusoidal endothelial cell survival according to
Rely in serum and endothelial growth factor, but the amplification environmental requirement serum-free of candidate stem cell and without endothelial cell growth
The factor will appear tire sinusoidal endothelial cell after co-culturing the short period so as to cause tire sinusoidal endothelial cell and candidate stem cell
Apoptosis.For this purpose, inventor is by E4orf1 channel genes tire sinusoidal endothelial cells so that tire sinusoidal endothelial cell can be in serum-free
With survive under conditions of endothelial growth factor and maintain self-characteristic, and need not move through mitomycin C or penetrate
The pretreatment of line can maintain the existing state not being proliferated.To build candidate stem cell microenvironment, hematopoiesis support stem cell
Amplification, and promote pluripotent stem cell to HSCs break up or directly reprogramming body cell acquisition HSCs.
According to an embodiment of the invention, this method further comprises：By first vector and carry the of GFP genes respectively
Two carriers are added jointly in the culture medium of tire sinusoidal endothelial cell.In order to observe conversion after cell, and by its with do not import target
The cell differentiation of gene, inventor attempt reporter gene importing tire sinusoidal endothelial cell.It is found by many experiments, with
The GFP genes of encoding green fluorescent protein are made as that in tracer channel genes tire sinusoidal endothelial cell, can not only play tracer
With nor affecting on the expression of E4orf1 genes.It is particularly suitable for promoting pluripotent stem cell to HSCs differentiation or directly reprogramming
Body cell distinguishes when obtaining HSCs with initiator cell.
According to an embodiment of the invention, first vector be packaged with the retrovirus of plasmid MSCV-N E4orf1, and
Anti- puromycin gene is carried on the plasmid.(tire hepatic sinusoidal endothelium is thin using Retroviral Transfer host cell by inventor
Born of the same parents) so that target gene (E4orf1 genes) enters host cell and is integrated into cellular genome, and insetion sequence is thin in host
It is expressed in born of the same parents, generates destination protein.Wherein, since host cell does not contain anti-puromycin gene, a certain concentration can not be resistant to
Puromycin.In turn, after the first vector for carrying anti-puromycin gene imports host cell, enable to host thin
Born of the same parents generate resistance, can be grown in the culture medium containing a certain concentration puromycin, and the whether is successfully imported to determine
One carrier.As a result, tire sinusoidal endothelial cell can survive and tie up under conditions of serum-free and endothelial growth factor
Self-characteristic is held, and the pretreatment for needing not move through mitomycin C or emitting radiation can maintain the survival shape not being proliferated
State.To build candidate stem cell microenvironment, the amplification of hematopoiesis support stem cell, and promote pluripotent stem cell to HSCs
Differentiation or directly reprogramming body cell obtain HSCs.
According to an embodiment of the invention, Second support is the retrovirus for being packaged with plasmid pMX-GFP.Inventor uses
Retroviral Transfer host cell so that target gene (GFP genes) enters host cell and is integrated into cellular genome,
Insetion sequence is expressed in host cell, generates destination protein.As a result, be convenient for observation conversion after cell, and by its with do not import
The cell differentiation of target gene, and E4orf1 gene expressions are not influenced.
According to an embodiment of the invention, first vector and the volume ratio of Second support are 1:1.Inventor passes through many experiments
Above-mentioned more excellent proportioning is obtained, the efficiency of cotransfection is best with this condition.
According to an embodiment of the invention, the puromycin containing 0.5 μ g/ml in the first culture medium, without containing serum and interior
Skin cell growth factor.Inventor has found that normal tire sinusoidal endothelial cell is free of anti-puromycin gene, therefore is containing 0.5 μ g/
It can not be grown in the culture medium of ml puromycins, in culture, second day starts with regard to gradual apoptosis.In turn, it is mould that anti-purine will be carried
The first vector and Second support of element import tire sinusoidal endothelial cell, enable to tire sinusoidal endothelial cell normal growth.Therefore,
The puromycin that 0.5 μ g/ml are added in the first culture medium, to play the role of screening transformed cells.
According to an embodiment of the invention, the first culture medium is EGM-2 basal mediums.As a result, tire hepatic sinusoidal endothelium is thin
Born of the same parents survive and maintain self-characteristic, to build candidate stem cell microenvironment, are conducive to the amplification of candidate stem cell, and promote more
Pluripotent stem cell breaks up to HSCs or directly reprogramming body cell obtains HSCs.
It will be appreciated to those of skill in the art that above for feature and advantage described in recombinant cell, it is same suitable
For the method for the Prepare restructuring cell, details are not described herein.
The method of hematopoietic stem cell expansion
In still another aspect of the invention, the present invention proposes a kind of method of hematopoietic stem cell expansion.It is according to the present invention
Embodiment, this method include：The method of Prepare restructuring cell obtains recombinant cell as described above；And by candidate stem cell
It is inoculated in the second culture medium containing recombinant cell, is cultivated, to make hematopoietic stem cell expansion.The recombination of the present invention is thin
Born of the same parents carry E4orf1 genes so that tire sinusoidal endothelial cell can be deposited under conditions of serum-free and endothelial growth factor
It is living and maintain self-characteristic, and the pretreatment for needing not move through mitomycin C or emitting radiation can maintain not to be proliferated
Existing state.To build candidate stem cell microenvironment, be conducive to candidate stem cell amplification, and promote pluripotent stem cell to
HSCs breaks up or directly reprogramming body cell obtains HSCs.
According to an embodiment of the invention, candidate stem cell CD34+Cell.CD34 is the important symbol of candidate stem cell,
In turn, inventor from the mononuclearcell of Cord blood by being isolated and purified with wherein CD34+Candidate stem cell.
According to an embodiment of the invention, serum and endothelial growth factor, the second culture are not contained in the second culture medium
Base is StemSpan culture mediums, contains 50ng/ml SCF (Stem cell factor), 50ng/ml Flt-3L (Fms-like
Tyrosine kinase 3ligand) and 50ng/ml TPO (Thrombopoietin).As a result, to realize candidate stem cell
Amplification and maintenance self-renewing, meanwhile, the existing state that tire sinusoidal endothelial cell can be maintained not to be proliferated.
It will be appreciated to those of skill in the art that feature described in method above for Prepare restructuring cell and excellent
Point, the method for being equally applicable to the hematopoietic stem cell expansion, details are not described herein.
Kit
In still another aspect of the invention, the present invention proposes a kind of kit.According to an embodiment of the invention, the kit
Including：Tire sinusoidal endothelial cell or recombinant cell noted earlier.It as a result, can using kit according to the ... of the embodiment of the present invention
Candidate stem cell microenvironment is built, is conducive to the amplification of candidate stem cell, and promotion pluripotent stem cell to HSCs differentiation or directly
It connects reprogramming body cell and obtains HSCs.
According to an embodiment of the invention, kit is for building in candidate stem cell microenvironment.
According to an embodiment of the invention, kit is used for amplifying candidate stem cell, maintains its self-renewing.
It will be appreciated to those of skill in the art that feature described in method above for hematopoietic stem cell expansion and
Advantage is equally applicable to the kit, and details are not described herein.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it is carried out according to technology or condition described in document in the art or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
1, human cord blood (Umbilical cord blood, UCB) CD34 is detached+Cell
Cord blood is transferred in sterile T75 Tissue Culture Flasks, erythrocyte sedimentation liquid is added by the one third of total volume, fills
Mixing, natural subsidence 20-40min is divided to be collected in upper plasma layer to 50ml centrifuge tubes with dropper, room temperature 2000rpm centrifugations
After 5min, 10ml PBS washed once, and cell is resuspended with 5ml PBS；Mononuclearcell is obtained with density-gradient centrifugation method
5ml peripheral blood lymphocytes separating liquids are added in (Mononuclear cells, MNCs) in 15ml centrifuge tubes, will with dropper
5ml cell suspensions are added slowly to along centrifugation tube wall on separating liquid liquid level at separating liquid ullage 1cm, and centrifuge tube is placed in
In horizontal centrifuge, 20min is centrifuged with deep low gear lifting speed room temperature 2000rpm, pipe inner cell suspension is divided into four layers after centrifugation, uses
Dropper draws canescence cloud cell and is placed on 50ml centrifuge tubes, and after addition PBS is washed 1 time, cell is resuspended to 1.5ml EP
CD34 sorting magnetic beads are added after 4 DEG C are protected from light rotation incubation 40min according to CD34Microbeads kit specifications, PBS in Guan Zhong
Washing 1 time, carries out CD34 in magnetic field+Cell sorting.
2, the preparation of E4orf1-GFP/HFLSECs feeder layers
(1) optimum medicine concentration of the HFLSECs of screening stable transfection MSCV-N E4orf1
VWF is also referred to as VIII factor Ⅷ related antigen, is a kind of high molecular weight protein poly for being synthesized and being secreted by vascular endothelial cell
Body participates in the formation of blood clotting and thrombus, therefore is often identified that the endothelium of in vitro culture is thin as characterization factor in vivo
Born of the same parents.Immunofluorescence results (Fig. 1) show that the vWF expression of the HFLSECs of original cuiture is positive.
HFLSECs is digested with 0.25% pancreatin, with 1 × 105/ hole is inoculated in 24 orifice plates and is cultivated, and after 16-24h, replaces
It is changed to the EGM-2 basal mediums containing 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 4 μ g/ml Puromycin respectively, last hole
HFLSECs still observes cell under the microscope as a control group with the EGM-2 base culture bases without Puromycin
Survival condition.The results show that under the condition of culture containing 0.5 μ g/ml puromycin, the original cuiture of script adherent growth
HFLSECs since the 2nd day be gradually rounded, gradually float in culture medium (Fig. 2) from the 3rd day, observed substantially not in 5 days
To the cell of adherent growth, and 1 μ g/ml, 2 μ g/ml, 4 μ g/ml puromycin condition of culture under, original cuiture
HFLSECs just all floatings from the 2nd day, drug effect is too strong, therefore, selects 0.5 μ g/ml puromycin dense for most suitable medicine sieve
Degree.
(2) HFLSECs fluidic cells sort after retrovirus packaging, cell transfecting, drug screening and transfection
Make retrovirus packaging plasmid MSCV-N E4orf1 and pMX-GFP respectively, and collects two kinds of venom.Wherein, it wraps
Can be seen that in retroviral producer cells after filling 20 hours has GFP expression, and expression rate is more than 90% (Fig. 3).Two kinds of venom are pressed
According to 1:1 ratio transfected HFLSECs simultaneously, and using 0.5 μ g/ml Puromycin pressurizations screening one week, then to carry out streaming thin
Born of the same parents analyze, and GFP positive rates are 90.5%, and GFP is obtained by airflow classification+Cell (Fig. 4 A), the E4orf1-GFP/ after sorting
HFLSECs can stablize amplification passage (Fig. 4 B) in the culture medium containing 0.5 μ g/ml Puromycin, be transferred to serum-free
EGM-2 in cultivated, remain to maintain survival and not be proliferated (Fig. 4 C), to obtain E4orf1-GFP/HFLSECs raising
Confluent monolayer cells.
FCM analysis is carried out to E4orf1-GFP/HFLSECs cells.The results are shown in Figure 5, E4orf1-GFP/
The positive rate of endothelial cell surface the mark CD144 and CD31 of HFLSECs are respectively 99% and 99.5%, and stem cell surface mark
Will CD117, progenitor endothelial cell surface mark CD133 and Blood cell surface phenotype CD45 are then hardly expressed, blood vessel endothelium life
Growth factor receptor body KDR expression rates are 69%.
3, CD34s of the E4orf1-GFP/HFLSECs as feeder layer culture human cord blood source+Cell
Separation obtains CD34 from UCB+Cell, with 5 × 104/ hole is inoculated in E4orf1-GFP/HFLSECs, with no feeding
Support layer suspension culture as a control group, with StemSpan culture mediums (SCF containing 50ng/ml, 50ng/ml Flt-3L, 50ng/
Ml TPO) continuously cultivate 15 days, when total number of cells are more than 1 × 106When, it needs cell being passaged on new feeder layer and continue
Culture.Fig. 6 shows in E4orf1-GFP/HFLSECs as in the amplification in vitro system of feeder layer, human cord blood source
CD34+15 days karyocyte sums of cell have expanded 360 times, and simple factor suspension culture group as a contrast only expands
165 times, the amplification efficiency of experimental group is 2.2 times of control group.
In the amplification in vitro system using E4orf1-GFP/HFLSECs as feeder layer, the CD34 in human cord blood source+
Cell still has the ability (Fig. 7) for being divided into different CFU, including erythroid burst colony formation unit in vitro after amplification
(BFU-E), colony-forming unit-erythroid (CFU-E), grain system colony forming unit (CFU-G), macronucleus system colony forming unit
(CFU-M), grain system and macrophage system colony forming unit (CFU-GM) and red system, grain system, macronucleus system, macrophage system mixing Colony forming
Unit (CFU-EGMM).
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (10)
1. purposes of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment.
2. purposes according to claim 1, which is characterized in that the tire sinusoidal endothelial cell is in the form of recombinant cell
It provides, the recombinant cell carries E4orf1 genes and optional GFP genes；
Optionally, the tire sinusoidal endothelial cell is used as feeder cells；
Optionally, the tire sinusoidal endothelial cell is for maintaining hematopoietic stem cell expansion；
Optionally, endothelial progenitor cells mark CD133, dryness mark CD117 and hematopoietic cell mark on the recombinant cell
The expression rate of CD45 is below 0.4%, and vascular endothelial growth factor receptor KDR expression rates are 55~80%, the mark of endothelial cell
Will CD144 and CD31 expression rate is all higher than 99%.
3. a kind of recombinant cell, which is characterized in that the recombinant cell is the tire sinusoidal endothelial cell for carrying E4orf1 genes.
4. recombinant cell according to claim 3, which is characterized in that the tire sinusoidal endothelial cell carries GFP genes；
Optionally, the recombinant cell is used as feeder cells.
5. a kind of method preparing the recombinant cell of claim 3 or 4, which is characterized in that including：
The first vector for carrying E4orf1 genes is added in the first culture medium containing the tire sinusoidal endothelial cell, is carried out
Culture, to obtain the recombinant cell.
6. according to the method described in claim 5, it is characterized in that, further comprising：
The first vector is added to the training of the tire sinusoidal endothelial cell jointly with the Second support for carrying GFP genes respectively
It supports in base,
Optionally, the first vector is to be packaged with the retrovirus of plasmid MSCV-N E4orf1, and taken on the plasmid
With anti-puromycin gene,
Optionally, the Second support is the retrovirus for being packaged with plasmid pMX-GFP；
Optionally, the first vector and the volume ratio of Second support are 1:1；
Optionally, the puromycin containing 0.5 μ g/ml in first culture medium, without containing serum and endothelial cell growth because
Son；
Optionally, first culture medium is EGM-2 basal mediums.
7. a kind of method of hematopoietic stem cell expansion, which is characterized in that including：
Recombinant cell is obtained according to the method for the Prepare restructuring cell of embodiment 5 or 6；And
Candidate stem cell is inoculated in the second culture medium containing the recombinant cell, is cultivated, to make the hematopoiesis
Expansion of stem cells.
8. the method according to the description of claim 7 is characterized in that the candidate stem cell is CD34+Cell；
Optionally, serum and endothelial growth factor are not contained in second culture medium, second culture medium is
StemSpan culture mediums contain 50ng/ml SCF, 50ng/ml Flt-3L and 50ng/ml TPO.
9. a kind of kit, which is characterized in that including：Tire sinusoidal endothelial cell or the recombinant cell of claim 3 or 4.
10. kit according to claim 9, which is characterized in that the kit is used to build the micro-loop of candidate stem cell
In border；
Optionally, the kit is for expanding the candidate stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810288645.8A CN108676777A (en) | 2018-04-03 | 2018-04-03 | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810288645.8A CN108676777A (en) | 2018-04-03 | 2018-04-03 | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108676777A true CN108676777A (en) | 2018-10-19 |
Family
ID=63800240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810288645.8A Pending CN108676777A (en) | 2018-04-03 | 2018-04-03 | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108676777A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643784A (en) * | 2012-03-29 | 2012-08-22 | 中国人民解放军第四军医大学 | Expansion system in vitro for hematopoietic stem/progenitor cell |
| US20140045260A1 (en) * | 2007-01-19 | 2014-02-13 | Cornell Research Foundation, Inc. | Methods and compositions for promoting survival and proliferation of endothelial cells and stimulating angiogenesis |
| CN105051184A (en) * | 2013-01-15 | 2015-11-11 | 康奈尔大学 | Reprogramming of human endothelium into hematopoietic multi-lineage progenitors by defined factors |
-
2018
- 2018-04-03 CN CN201810288645.8A patent/CN108676777A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140045260A1 (en) * | 2007-01-19 | 2014-02-13 | Cornell Research Foundation, Inc. | Methods and compositions for promoting survival and proliferation of endothelial cells and stimulating angiogenesis |
| CN102643784A (en) * | 2012-03-29 | 2012-08-22 | 中国人民解放军第四军医大学 | Expansion system in vitro for hematopoietic stem/progenitor cell |
| CN105051184A (en) * | 2013-01-15 | 2015-11-11 | 康奈尔大学 | Reprogramming of human endothelium into hematopoietic multi-lineage progenitors by defined factors |
Non-Patent Citations (4)
| Title |
|---|
| BUTLER等: "Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells", 《CELL STEM CELL》 * |
| OLGA WITTIG等: "Liver Sinusoidal Endothelial Cells Promote B Lymphopoiesis From Primitive Hematopoietic Cells", 《STEM CELLS AND DEVELOPMENT》 * |
| SEANDEL等: "Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene", 《PNAS》 * |
| 余艳等: "造血干细胞体外扩增方法研究进展", 《医学综述》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Knezevic et al. | Engineering blood and lymphatic microvascular networks in fibrin matrices | |
| Trkov et al. | Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions | |
| CN103937743B (en) | A kind of method that candidate stem cell is obtained using three-dimensional inducible system | |
| Bagley et al. | Extended culture of multipotent hematopoietic progenitors without cytokine augmentation in a novel three-dimensional device | |
| CN101857854A (en) | Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof | |
| CN106834224A (en) | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell | |
| CN101821383A (en) | A kind of primary mesenchymal stem cells and application thereof that is used for substratum, method and the acquisition of the external mass-producing cultivation of human adult primary mesenchymal stem cells | |
| CN105378069A (en) | Human extensively self-renewing erythroblasts (ESRE) | |
| Di Buduo et al. | Latest culture techniques: cracking the secrets of bone marrow to mass-produce erythrocytes and platelets ex vivo | |
| Bessy et al. | Bioengineering the bone marrow vascular niche | |
| CN108291196A (en) | Express the endothelial cell of the engineering of ETS transcription factors | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN113308434B (en) | Construction method of endothelial cell and pericyte co-culture model for researching tube formation | |
| JP2014060991A (en) | Method of culturing stem cells using inner lumen of porous hollow fiber | |
| CN108220243B (en) | A kind of multipotential stem cell and its T cell and application of differentiation | |
| CN107674858A (en) | The isolation medium and separation method of marrow endothelial progenitor cell | |
| CN106754650B (en) | A kind of endothelial progenitor cells cultural method of derived from bone marrow | |
| CN104630135B (en) | Method for preparing hepatic stem cells on large scale and application thereof | |
| CN101063110B (en) | Culture medium and method for architectonic stem cell | |
| CN115927164B (en) | Culture method and application of vascularized tumor organoids | |
| Butler et al. | Generation of a vascular niche for studying stem cell homeostasis | |
| CN109182268B (en) | Application of umbilical artery endothelial cells in constructing microenvironment of blood stem cells | |
| CN108165529A (en) | Central memory T cell body and its outer cultural method | |
| CN108676777A (en) | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation | |
| CN104195107B (en) | Purposes of the microcapsule bubble in induction stem cell macronucleus differentiation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220105 Address after: 100850 No. 27 Taiping Road, Beijing, Haidian District Applicant after: ACADEMY OF MILITARY MEDICAL SCIENCES Applicant after: South China Institute of biomedicine Address before: No.1, helix 4 road, International Biological Island, Guangzhou, Guangdong 510200 Applicant before: SOUTH CHINA INSTITUTE OF BIOMEDICINE |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181019 |
|
| RJ01 | Rejection of invention patent application after publication |