CN106754718A - A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium - Google Patents
A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium Download PDFInfo
- Publication number
- CN106754718A CN106754718A CN201611181167.8A CN201611181167A CN106754718A CN 106754718 A CN106754718 A CN 106754718A CN 201611181167 A CN201611181167 A CN 201611181167A CN 106754718 A CN106754718 A CN 106754718A
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cell
- culture
- human embryo
- final concentration
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
Abstract
Break up to the method for retinal pigment epithelium the invention discloses a kind of human embryo stem cell slewing, ES cell differentiation be ectodermic NSC and by the neural stem cell differentiating stage into retinal stem cells use Noggin albumen, DKK 1, bFGF and niacinamide promote the generation of NSC and retinal stem cells, it is RPE cells then to use activin A and SU5402 to promote the quick differentiation and maturation of retinal stem cells, finally promote RPE cell fast-ripenins using maturation medium, whole process can be completed in 30 days, not only it is efficiently but also quick.
Description
Technical field
The present invention relates to a kind of cell culture processes, and in particular to a kind of human embryo stem cell slewing is broken up to view
The method of membranochromic pigments epithelial cell.
Background technology
Retinal pigment epithelium (Retinal pigment epithelial cell, abbreviation RPE cell) is one layer
It is close to the chromatophore outside retinal visual nerve.The RPE cells of fusion are presented individual layer hexagon, cell under the microscope
It is contained within intensive pigment granule.RPE cells have various kinds of cell function, for example, absorbing light, epithelial transhipment, ion delays
Punching, vision loop, cell phagocytosis, secretion and immunological regulation, for maintain retina on various Visual Neuron cells it is normal
Function plays an important role1-2.Wherein important function has：1) epithelial transhipment：RPE cells form blood retina barrier
(Blood retina barrier), it is combined with the cell tight outside double-sided surface, and makes retinochrome independently of outside
The influence of system, this has important meaning to the high selectivity substance transportation in eye precision control environment and nerve signal transmission
Justice, meanwhile, RPE cells provide nutrition for photosensory cell, regulate and control ion concentration, exclude water and other metabolites；2) for sense
The cytophagy (Phagocytosis) of the outer section (Photoreceptor outer segment) of photo-cell：Photosensory cell
Photooxidation stimulation is constantly exposed to, can necessarily be damaged, the outer section that RPE cells will be constantly damaged by cytophagy
Swallow and digest, to maintain the normal function of photosensory cell；3) secrete cytokines：RPE cells can secrete it is substantial amounts of it is various because
Son and signaling molecule to be exchanged with the tissue implementation around it, e.g., fibroblast growth factor, transforming growth factor-β, class pancreas islet
Plain growth factor-1, platelet derived growth factor, VEGF and the pigment epithelium-derived factor etc., these letters
Number molecule has important physiopathology meaning.If feature lesion occur in RPE cells, will directly affect relative
Retina, it will usually cause inpairment of vision, or even blind disease.Most common optical disorders have age-related macular degeneration and retinal pigment
Denaturation3。
Age-related macular degeneration (Age related macular degeneration, abbreviation AMD) is one kind and age phase
The ophthalmology disease of pass, its incidence of disease is high, is common in elderly population, be more than 55 years old the elderly's low vision blinding it is main
Reason, has a strong impact on life of elderly person quality.In European and American areas, more than the 55 years old illness rate of crowd is 1.63%, over-65s
Illness rate about 16%, the illness rate of more than 75 years old is close to 30%4-5.China is with aging population and hypertension and hyperglycaemia
The increase of patient populations, AMD numbers of patients also gradually rise.The World Health Organization once delivered report, it is indicated that the whole world is at least
8000000 people are subjected to the pain that eyesight major injury brings due to advanced senile macular degeneration.
AMD pathogenesis is that human vision is imaged most sharp macular area generation degenerative lesion.Occurs decline first
It is the retinal pigment epithelium below retina, is followed by the retina sense with retinal pigment epithelium direct neighbor
There is apoptosis in photo-cell, and the eyesight for causing the visual field to hit exactly significantly declines, or even blindness.AMD is according to different points of its clinical manifestation
It is atrophic type (also known as dryness) and exudative type (also known as moist) amphitypy, dryness is mainly under RPE cells piles up toxicity precipitation etc. and draw
Play RPE cells and photoperiod sensitivity gene；Moist predominantly CNV is formed, and is invaded under retina, causes macular area
Bleeding simultaneously forms disciform detachment, ultimately forms plate-like scar, causes the serious loss of central region eyesight.AMD is definite to cause a disease
Reason is still unclear, in terms of some researchs show that main pathogenic has E＆H3,6。
Serious blinding corresponding with AMD is a lack of effective treatment method.Anti- new life can be used now to moist AMD
Blood vessel drug eluting is treated.But for dryness AMD, at present without any effective treatment means.Some clinical researches show to make
Macula lutea can be delayed to be formed with B B-complex, but final blindness is still inevitable.
HESC (Human embryonic stem cell, abbreviation hESC) is dry thin as a kind of totipotency
Born of the same parents (Pluripotent stem cell), with two kinds of other cells without characteristic：One can be oneself of unlimited merisis
My updating ability；Two can be the potential for being divided into the every other cell line of human body.Under specific condition of in vitro culture, can be with
HESC is broken up to the body cell of difference in functionality, and then form different tissue even organs.What embryonic stem cell had
These potential have very strong application prospect to medical field.Modern medicine is still felt simply helpless many human diseases, this kind of disease
The pathogenesis of disease are mostly because lose one or more functioning cells, and human body cannot regenerate the cell that these lose.
If hESC is divided into cell that these lose and patient is transplanted back, just there is a strong possibility cures these completely does not have originally
Any desired disease, these diseases include：Type i diabetes, senile dementia, amyotrophic lateral sclerosis, epidermis
Dissolving water Blisters diseases, heart failure, miocardial infarction, ischemic cardiac myositis and sickle anaemia etc.7.While embryonic stem cell is also
Can combine gene therapy, organizational project and drug development etc. carry out medical treatment or research application, therefore embryonic stem cell medical treatment
Application prospect is very wide, there is huge social demand and market potential.
The scientist of current Britain, the U.S., Japan and South Korea is attempting the RPE cells (hESC- of transplanting hESC differentiation
RPE) the RPE cells failed to the retina substitution of patient, to treat AMD.The scientist of South Korea CHA University with it is beautiful
Four patients AMD were treated, wherein three people show by the Ocata Therapeutics cooperations of drugmaker of state in 2013
Go out therapeutic effect, any side effect is not found after long-term observation8.The scientist of Japan Chemical research institute and the municipal central city in Kobe
People hospital cooperates, and induction totipotency stem cell (Induced pluripotent stem cell, abbreviation were used in 2014
IPSC) the RPE cells of differentiation implement treatment to patient AMD14.British scientist has been set up with hESC-RPE treatments AMD's
London Project to Cure Blindness, there are Moorfields Eye in the research of participation and commercial undertaking
Hospital、UCL Institute of Ophthalmology、National Institute for Health
Research and Pfizer company, successfully implement transfer operation, and plan in September, 2015 to patient AMD
10 patients are treated in 18 months15.California, USA regenerative medicine research institute has been subsidized with hESC-RPE treatments AMD's
There are University of Southern in California Project to Cure Blindness, the research institution of participation
California, UC Santa Barbara, Caltech, City of Hope and Cedar Sinai Hospital, with
Begin preparing within 2016 clinical phase experiment.
In the stem-cell therapy to AMD, the differentiation technique of RPE cells has consequence.The differentiation effect of RPE cells
Whether the height of rate, the yield of different batches RPE cells is stablized, and whether every proterties of RPE cells reaches normal level decision
The quality of subsequent therapeutic effect.The method of the Spontaneous Differentiation RPE cells that tradition is used needs longer time, has in recent years
Accelerate RPE cell differentiations using the method for directed differentiation, but differentiation efficiency is still relatively low, the RPE cells after breaking up in addition
Maturation still need for a long time9-13, these shortcomings are all unfavorable for the clinical medicine application of RPE cells.
The content of the invention
In view of this, break up to retinal pigment epithelium the invention provides a kind of human embryo stem cell slewing
Method, the method can be in 30 days by human embryo stem cell induction differentiation to ripe retinal pigment epithelium, tool
Have the advantages that rapidly and efficiently.
The technical scheme that the present invention takes is as follows：
1. a kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium, including following step
Suddenly：
(1) human embryo stem cell culture is carried out first, culture medium is discarded when cell growth to 80% fusion, add differentiation
Basal medium, while adding the Noggin factors of final concentration 50ng/mL, the DKK-1 factors of final concentration 10ng/mL and final concentration
The niacinamide of 10mM continues to cultivate；Based on 500mL, component and each component content are the differentiation basal medium：
1 × DMEM/F12 culture medium 480mL,Supplement 10mL, 100 × N-2Supplement
5mL, 100 × MEM nonessential amino acid solution 5mL；The volume ratio of DMEM and F12 is 1: 1 in the DMEM/F12 culture mediums；
(2) old culture medium is discarded within the 3rd day after breaking up, the differentiation basal medium for more renewing, while adding final concentration 10ng/
The Noggin factors of mL, the DKK-1 factors of final concentration 10ng/mL, the niacinamide of final concentration 10mM and final concentration 5ng/mL's
BFGF continues to cultivate；
(3) old culture medium is discarded within the 5th day after breaking up, the differentiation basal medium for more renewing, while adding final concentration 10ng/
The Noggin factors of mL, the DKK-1 factors of final concentration 10ng/mL, the niacinamide of final concentration 10mM and final concentration 5ng/mL's
BFGF continues to cultivate；
(4) the 7th~8 day after breaking up, when occurring 5~15% NSC in cell culture, old culture is discarded
Base, the differentiation basal medium for more renewing, while adding the activin A of final concentration 100ng/mL and the SU5402 of 10 μM of final concentration
Continue to cultivate to after breaking up the 14th day, every 2 days of period changed a subculture；
(5) the 15th day part cell is differentiated to retinal pigment epithelium after breaking up, and discards old culture medium, changes
Maturation medium continues large area pigment patch occur in culture to cell culture；
Based on 500mL, component and each component content are the maturation medium：1 × IMDM culture medium 494.5mL,Supplement 5mL, the lipid concentrate that the chemical composition diluted by 1: 1000 volume ratio determines
(Chemically defined lipid concentrate)0.5mL。
Preferably, the vessel bottom of human embryo stem cell culture is coated with one layer of Matrigel in the step (1).
Preferably, using the medium culture human embryo stem cells of Essential 8 to 80% fusion in the step (1),
Culture medium is discarded, basal medium is broken up with being added after DMEM/F12 culture medium washed cells.
Preferably, when human embryo stem cell grows to 80% fusion in the step (1), remove by hand differentiated miscellaneous thin
Born of the same parents.
Preferably, first with DMEM/F12 culture mediums to thin before the differentiation basal medium for more renewing in the step (4)
Born of the same parents are washed.
Preferably, the human embryo stem cell is H9 cell lines.
The 2.Noggin factors break up answering into retinal pigment epithelium in promotion human embryo stem cell slewing
With.
3. niacinamide is in the application for promoting human embryo stem cell slewing to break up into retinal pigment epithelium.
4.SU5402 is in the application for promoting human embryo stem cell slewing to break up into retinal pigment epithelium.
In the present invention, Noggin factors concentration may range from 10~100ng/mL, DKK-1 factor concentrations
May range from 5~20ng/mL, niacinamide concentration may range from 5~15ng/mL, bFGF concentrations scope can be with
It is 5~10ng/mL, activin A concentration may range from 80~120ng/mL, SU5402 factor concentrations scope can be with
It is 10~20 μM.
Noggin is a kind of polypeptide protein secreted by notochord, is important regulating and controlling necessary to nervous system and skeleton development
The factor, animal model confirms that missing Noggin genes can cause the defect of the nervous systems such as spinal nerve pipe deformity.Noggin eggs
White Main Function approach is that, by combining simultaneously Developing restraint factor TGF-β family member, such as BMP4 factors are various to play
Regulating and controlling effect.Adding Noggin can effectively improve ES cell differentiation to NSC and the efficiency of retinal stem cells.
DKK-1 is the suppression molecule of WNT signal paths, and main effect model is to suppress the frizzled receptor on WNT paths
LRP6, reduces the level of beta chain albumen and improves the expression of OCT4.DKK-1 is in head, the development of heart and upper limbs
Play an important role.Zooscopy shows that DKK-1 expression deletions can cause a series of neurological function deficits, such as eye development missing,
Allotriosmia, forebrain and midbrain developmental defect etc..
Fibroblast growth factor (FGF) is the multi-functional growth factor with extensive bioactivity and physiological function,
Cell growth and differentiation and function all have an impact.Especially in nervous system, bFGF is widely present and has to neuron
Survive and promote reparation and the palingenesis of growth and injured neurons.In vitro in cell culture, bFGF and epidermis are given birth to
The factor (EGF) long is to induce and maintain medium component necessary to NSC normal growth.
Niacinamide (Nicotinamide) is nicotinic acid (vitamin B3) acid amides.In cell, nicotinic acid be used to synthesize cigarette
Acid amides adenine-dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP), and the transition pathway of niacinamide with
The approach of nicotinic acid is closely similar, so the precursor that can synthesize as NAD.Experiment in vitro confirms that niacinamide has protection to nerve cell
Effect, can improve neuronal cell survival rate.
Activin A (Activin A) belongs to the member of growth factor TGF-β family, mainly by various bodies of gland, such as reproduction
The secreting, expressings such as gland, pituitary, also can high level expression when being healed after skin histology injury.Its effect is chiefly to facilitate various non-
The form generation of nerve fiber.Form to the epithelial tissue belonging to RPE cells is formed, and polarity and physiological function have promotion to make
With.
SU5402 (2- [(1,2- dihydro-2-oxo -3H- indoles -3- subunits) methyl] -4- methyl isophthalic acid H- pyrroles -3- third
Acid) it is a kind of chemical small molecule, to FGF receptor, vegf receptor and pdgf receptor have inhibitory action.BFGF is to induce and maintain god
Through medium component necessary to stem cell normal growth, SU5402 is added to suppress the differentiation of retinal neuronal cell,
So as to improve RPE cell differentiation efficiency.
As shown in figure 1, ES cell differentiation can be roughly divided into three phases to the process of RPE cells, one is that embryo does
Cell differentiation is ectodermic NSC, and two is into retinal stem cells, finally by retina by neural stem cell differentiating
Stem cell is divided into two kinds of different tissues --- the main retina being made up of various neuronal cells and individual layer polarity
RPE cells.Because RPE cell deriveds are in optic nerve tissue, but it is not neuronal cell, so mistake of the RPE cells in differentiation
The regulation and control being subject in journey are complex, and pre-differentiation stage needs specific regulatory factor to promote nerve fiber to be formed, and differentiation stage is needed
Other regulatory factors are wanted to suppress the formation of nerve fiber.Therefore, the needs when promoting human embryo stem cell to break up to RPE cells
The various signaling molecules and growth factor fine-tuned in directed differentiation of fixed place and time.
The present invention promotes NSC in above-mentioned the first two stage using Noggin albumen, DKK-1, bFGF and niacinamide
And the generation of retinal stem cells, it is RPE then to use activin A and SU5402 to promote the quick differentiation and maturation of retinal stem cells
Cell, finally promotes RPE cell fast-ripenins using maturation medium, and whole process can be completed in 30 days, not only efficiently but also
Quickly.
Brief description of the drawings
In order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art, below will be to specific
The accompanying drawing to be used needed for implementation method or description of the prior art is briefly described.
Fig. 1 present invention signaling molecule used and growth factor are in the work for promoting human embryo stem cell to break up into RPE cells
With and schematic diagram of mechanism；
Fig. 2 is the cellular morphology of the first stage (1-7 days) of human embryo stem cell H9 directed differentiations；First day (Day 1),
H9 cells are presented typical undifferentiated embryonic stem cell group form under low power lens (4 ×)；It is visible thin under high power lens (10 ×)
Born of the same parents group is cell monolayer；4th day (Day 4), population of cells breaks up, population of cells's form significant change under low power lens,
Group edge becomes rough, and group's shape is no longer circular, and multi-layer cellular heap occurs in visible cell group center under high power lens
Product, the cell shape at group edge is changed into elongated；7th day (Day 7), population of cells's form is further change in, can under low power lens
See that whole group is changed into multi-layer cellular accumulation, visible a large amount of typical NSC morphological features under high power lens：It is rose-shaped
Structure (neural rosette)；
Fig. 3 is the cellular morphology of the second stage (the 15th day) of human embryo stem cell H9 directed differentiations；A, cell culture
There is the small patch of a small amount of pigment；Pigment patch (mature RPE cells) under B, low power lens；Pigmented spots under C, high power lens
Block (mature RPE cells)；Non-pigment patch (immature RPE cells) under D, low power lens；It is colourless under E, high power lens
Plain patch (immature RPE cells), the cell outline of these immature RPE cells presented hexagon is aobvious in difference
Under micro mirror, cell edges are rendered as high brightness；
The cellular morphology of the phase III (the 30th day) of Fig. 4 human embryo stem cell H9 directed differentiations；A, cell culture goes out
Existing a large amount of pigment patches；Pigment patch (mature RPE cells) under B, low power lens；Pigment patch under C, high power lens is (
Ripe RPE cells)；Non-pigment patch (immature RPE cells) under D, low power lens；Non-pigment patch under E, high power lens
(immature RPE cells)；
The cellular morphology of last (the 60th day) of Fig. 5 human embryo stem cell H9 directed differentiations；A, cell culture occurs big
Amount pigment patch；Pigment patch (mature RPE cells) under B, low power lens；Pigment patch under C, high power lens is (ripe
RPE cells).
Specific embodiment
The embodiment of technical solution of the present invention is described in detail below in conjunction with accompanying drawing.
Human embryo stem cell used of the invention is H9 cell lines；The differentiation basal medium based on 500mL, component and each
Constituent content is：
1 × DMEM/F12 culture medium 480mL,Supplement (abbreviation B27) 10mL, 100 × N-2
Supplement (abbreviation N2) 5mL, 100 × MEM nonessential amino acid solutions (MEM Non-Essential Amino Acids
Solution, NEAA) 5mL；The volume ratio of DMEM and F12 is 1: 1 in the DMEM/F12 culture mediums；
Based on 500mL, component and each component content are the maturation medium：1 × IMDM culture medium 494.5mL,Supplement 5mL, the lipid concentrate that the chemical composition diluted by 1: 1000 volume ratio determines
(Chemically defined lipid concentrate, match is silent to fly, production number 11905031) 0.5mL.
A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium, comprises the following steps：
(1) Matrigel (production of Corning companies) covering cell culture with six orifice plates, use Essential
8 culture mediums carry out human embryo stem cell H9 cell line cultures, when cell growth to 80% fusion, discard culture medium, use DMEM/
F12 culture medium washed cells culture once after, differentiation basal medium 2mL is added per hole, while adding final concentration 50ng/mL
The Noggin factors, the DKK-1 factors of final concentration 10ng/mL and final concentration 10mM niacinamide continue cultivate；
It is noted that when human embryo stem cell grows to 80% fusion, needing first to remove differentiated heteroproteose cell by hand；Just
The human embryo stem cell cultivated in the case of often can all have a small amount of differentiated cell to mix in the undifferentiated cell for occupying the majority, just
After often culture 6-8 days, when cell fusion degree reaches 80% or more, the quantity of the differentiated cell mixed in cell culture product
Comparing ratio high (about 5-20%) will be reached, if directly carrying out RPE cell directional differentiation using the cell culture, is led to
Chang Buhui obtains yield high, because differentiated cell belongs to random differentiation, there is a large amount of other types of body cells, its
In some cell types, such as fibroblast and endothelial cell have very strong growth vigor, if not differentiation initial stage row
Except these quick heteroproteose cells of growth, then the ratio of RPE cells will be very low in final noble cells product, so opening
Before beginning is broken up, it should carry out microscope detection first, judge the ratio of differentiated cell, if differentiated cell proportion is super
5% is crossed, then needs to carry out removing differentiated cell by hand under surgical operation microscope；When removing by hand, should be under surgical operation microscope
Using P10 pipettors and P10 pipette tips, rule along neoblast group edge, then reuse pipette tips by scribe area
Differentiated cell shovel is removed；Cell is removed after the completion of operation, should wash culture hole using fresh culture；
(2) discard old culture medium within the 3rd day after breaking up, differentiation basal medium 2mL is added per hole, while adding final concentration
The Noggin factors of 10ng/mL, the DKK-1 factors of final concentration 10ng/mL, the niacinamide of final concentration 10mM and final concentration 5ng/mL
BFGF continue cultivate；
(3) discard old culture medium within the 5th day after breaking up, differentiation basal medium 2mL is added per hole, while adding final concentration
The Noggin factors of 10ng/mL, the DKK-1 factors of final concentration 10ng/mL, the niacinamide of final concentration 10mM and final concentration 5ng/mL
BFGF continue cultivate；
(4) occurs 5~15% NSC the 7th day after breaking up, in cell culture, embryonic stem cell has been induced
Differentiation removes old culture medium to NSC and retinal stem cells (see Fig. 2), be washed once with DMEM/F12 culture mediums
After cell culture, the differentiation basal medium for more renewing adds 2mL per hole, while adding the activation of final concentration 100ng/mL
The SU5402 of 10 μM of plain A and final concentration continues to cultivate to after breaking up the 14th day, and every 2 days of period changed a subculture；
(5) there is the small patch of pigment of light brown in the 15th day part cell culture after breaking up, and shows had part thin
Born of the same parents break up to RPE cells (see Fig. 3)；After removing old culture medium, after being washed once using RPE maturation mediums, RPE is added per hole
Maturation medium 2mL continues to cultivate, and every 2 days change a fresh culture, until occurring large area in cell culture
Pigment patch (about at the 45-60 days, seeing Fig. 4 and Fig. 5)；
(6) treat that 50% or more dark-brown pigment patch occurs in cell culture, you can isolate and purify RPE cells, can adopt
Isolation and purification method has two kinds：First, under surgical operation microscope, larger pigment patch is cut and is received using surgical scissors
Collection；2nd, using the enzymic digestion of many wheels, after the cell of other species is removed respectively, RPE cells are regathered；Use hand sample or enzyme
After the method for digestion isolates and purifies retinal pigment epithelium, the cell of precipitation is inoculated in RPE maturation cultures by centrifugation
In base, 37 DEG C, 5%CO are put2Cultivated in saturated humidity incubator；
(7) 1-3 is taken for retinal pigment epithelium identification of cell form and molecular labeling is determined, it was demonstrated that culture is tool
There is the retinal pigment epithelium of normal characteristics.
In incubation, it should be noted that the signaling molecule and growth factor of directed differentiation addition in first day and the 3rd day
The difference of concentration, because Noggin is the important regulating and controlling factor necessary to nervous system and skeleton development, so fixed in RPE cells
Noggin must be added to the early stage of differentiation to improve the efficiency of nerve fiber differentiation, but if be always maintained at high concentration
Noggin is then possible to improve the ratio of skeletal cell differentiation, so at first day of directed differentiation and second day, using highly concentrated
The Noggin of degree then reduces the ratio of skeletal cell differentiation to establish the direction of differentiation using the Noggin of low concentration.
Fibroblast growth factor (FGF) be induce with maintain NSC normal growth necessary to culture medium into
Part, but bFGF is also the necessary regulatory factor for maintaining embryonic stem cell morphological function, if added within first day in directed differentiation
Entering bFGF then can significantly extend the time of differentiation, be unfavorable for extensive industrialized production rapidly and efficiently, so in directed differentiation
First day and second day, bFGF is added without to promote embryonic stem cell to rapidly enter differential period, treat the 3rd day, add
BFGF induced nerve stem cells break up.
The change in concentration of the both the above factor has vital influence to efficient slewing differentiation RPE cells, if
Correctly can not add according to quantity on time, differentiation efficiency can be subject to extreme influence.
At the 5-7 days of directed differentiation, there is a small amount of rosettes structure (neural rosette, god in cell culture
Through the representative configuration feature of stem cell) after, should change in time plus Activin A and SU5402.
In (the 1-6 days) factor groups (Noggin, DKK-1, bFGF and Nicotinamide) of addition of directed differentiation early stage
With the effect that strong promotion nerve fiber is broken up, but because required final cell product RPE cells are not neural thin
Born of the same parents, so the rapid conversion differentiation direction after NSC and retinal stem cells occur is needed, to improve dividing for RPE cells
Change efficiency.The opportunity of conversion differentiation direction (addition Activin A and SU5402) is particularly important, according to experiment experience, when whole
When a small amount of (5-15%) rosettes structure occurs in cell culture, the best opportunity of Activin A and SU5402 is as added.
If rosettes structure ratio is higher than 25%, the differentiation direction of cell then biases toward nerve fiber, and a large amount of final cells are produced
Thing is then retinal neuronal cell, even brain neuron cell.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme, it all should cover in the middle of the scope of claim of the invention and specification.
Prior art literature
1.Strauss, O.The retinal pigment epithelium in visual function.Physiol
Rev 85,845-881 (2005)
2.Bok, D.The retinal pigment epithelium：a versatile partner in
Vision.J Cell Sci Suppl 17,189-195 (1993)
3.Zarbin, M.A.Current concepts in the pathogenesis of age-related
Macular degeneration.Arch Ophthalmol 122,598-614 (2004)
4.Jager, R.D., Mieler, W.F.＆Miller, J.W.Age-related macular
Degeneration.N Engl J Med 358,2606-2617 (2008)
5.de Jong, P.T.Age-related macular degeneration.N Engl J Med 355,1474-
1485(2006).
6.Zajac-Pytrus, H.M., Pilecka, A., Turno-Krecicka, A., Adamiec-Mroczek, J.＆
Misiuk-Hojlo, M.The Dry Form of Age-Related Macular Degeneration (AMD)：The
Current Concepts of Pathogenesis and Prospects for Treatment.Adv Clin Exp Med
24,1099-1104 (2015)
7.Strauer, B.E.＆Kornowski, R.Stem cell therapy in
Perspective.Circulation 107,929-934 (2003)
8.Song, W.K., et al.Treatment of macular degeneration using embryonic
stem cell-derived retinal pigment epithelium：preliminary results in Asian
Patients.Stem Cell Reports 4,860-872 (2015)
9.Klimanskaya, I., et al.Derivation and comparative assessment of
retinal pigment epithelium from human embryonic stem cells using
Transcriptomics.Cloning Stem Cells 6,217-245 (2004)
10.Buchholz, D.E., et al.Derivation of functional retinal pigmented
Epithelium from induced pluripotent stem cells.Stem Cells 27,2427-2434 (2009)
11.Hirami, Y., et al.Generation of retinal cells from mouse and human
Induced pluripotent stem cells.Neurosci Lett 458,126-131 (2009)
12.Buchholz, D.E., et al.Rapid and efficient directed differentiation
of human pluripotent stem cells into retinal pigmented epithelium.Stem Cells
Transl Med 2,384-393 (2013)
13.Leach, L.L., Buchholz, D.E., Nadar, V.P., Lowenstein, S.E.＆Clegg,
D.O.Canonical/beta-catenin Wnt pathway activation improves retinal pigmented
epithelium derivation from human embryonic stem cells.Invest Ophthalmol Vis
Sci 56,1002-1013 (2015)
14.http：//www.nature.com/news/next-generation-stem-cells-cleared-for-
human-trial-1.15897
15.http：//www.moorfields.nhs.uk/news/new-trial-wet-amd.
Claims (9)
1. a kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium, it is characterised in that including
Following steps：
(1) human embryo stem cell culture is carried out first, culture medium is discarded when cell growth to 80% fusion, add differentiation basis
Culture medium, while adding the Noggin factors of final concentration 50ng/mL, the DKK-1 factors of final concentration 10ng/mL and final concentration 10mM
Niacinamide continue cultivate；Based on 500mL, component and each component content are the differentiation basal medium：
1 × DMEM/F12 culture medium 480mL,Supplement 10mL, 100 × N-2Supplement 5mL,
100 × MEM nonessential amino acid solutions 5mL；The volume ratio of DMEM and F12 is 1: 1 in the DMEM/F12 culture mediums；
(2) old culture medium is discarded within the 3rd day after breaking up, the differentiation basal medium for more renewing, while adding final concentration 10ng/mL's
The Noggin factors, the DKK-1 factors, the niacinamide of final concentration 10mM and final concentration 5ng/mL of final concentration 10ng/mL bFGF after
Continuous culture；
(3) old culture medium is discarded within the 5th day after breaking up, the differentiation basal medium for more renewing, while adding final concentration 10ng/mL's
The Noggin factors, the DKK-1 factors, the niacinamide of final concentration 10mM and final concentration 5ng/mL of final concentration 10ng/mL bFGF after
Continuous culture；
(4) the 7th~8 day after breaking up, when occurring 5~15% NSC in cell culture, old culture medium is discarded, more
The differentiation basal medium for renewing, while adding the activin A of final concentration 100ng/mL and the SU5402 of 10 μM of final concentration to continue
To after breaking up the 14th day, every 2 days of period changed a subculture for culture；
(5) the 15th day part cell is differentiated to retinal pigment epithelium after breaking up, and discards old culture medium, changes ripe training
Support base and continue large area pigment patch occur in culture to cell culture；
Based on 500mL, component and each component content are the maturation medium：1 × IMDM culture medium 494.5mL,Supplement 5mL, the lipid concentrate 0.5mL that the chemical composition diluted by 1: 1000 volume ratio determines.
2. a kind of human embryo stem cell slewing according to claim 1 is broken up to the side of retinal pigment epithelium
Method, it is characterised in that the vessel bottom of human embryo stem cell culture is coated with one layer of Matrigel in the step (1).
3. a kind of human embryo stem cell slewing according to claim 1 is broken up to the side of retinal pigment epithelium
Method, it is characterised in that using the medium culture human embryo stem cells of Essential 8 to 80% fusion in the step (1), abandon
Culture medium is removed, basal medium is broken up with being added after DMEM/F12 culture medium washed cells.
4. a kind of human embryo stem cell slewing according to claim 1 is broken up to the side of retinal pigment epithelium
Method, it is characterised in that when human embryo stem cell grows to 80% fusion in the step (1), removes differentiated miscellaneous thin by hand
Born of the same parents.
5. a kind of human embryo stem cell slewing according to claim 1 is broken up to the side of retinal pigment epithelium
Method, it is characterised in that first with DMEM/F12 culture mediums to cell before the differentiation basal medium more renewed in the step (4)
Washed.
6. a kind of human embryo stem cell slewing according to any one of Claims 1 to 5 is broken up to retinal pigment
The method of chrotoplast, it is characterised in that the human embryo stem cell is H9 cell lines.
The 7.Noggin factors are in the application for promoting human embryo stem cell slewing to break up into retinal pigment epithelium.
8. niacinamide is in the application for promoting human embryo stem cell slewing to break up into retinal pigment epithelium.
9.SU5402 is in the application for promoting human embryo stem cell slewing to break up into retinal pigment epithelium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611181167.8A CN106754718A (en) | 2016-12-19 | 2016-12-19 | A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611181167.8A CN106754718A (en) | 2016-12-19 | 2016-12-19 | A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106754718A true CN106754718A (en) | 2017-05-31 |
Family
ID=58890891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611181167.8A Pending CN106754718A (en) | 2016-12-19 | 2016-12-19 | A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754718A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906178A (en) * | 2017-04-07 | 2017-06-30 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Cultivate the application in the composition and its holding epithelioid cell's form of epithelial cell |
| CN108531443A (en) * | 2018-04-04 | 2018-09-14 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | The method that small molecule inducing pluripotent stem cells are divided into retinal pigment epithelium |
| CN109136184A (en) * | 2018-07-16 | 2019-01-04 | 同济大学 | The method that induction people's multipotent stem cells are divided into RPE cell |
| CN110628696A (en) * | 2019-08-28 | 2019-12-31 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
| CN113549596A (en) * | 2021-08-04 | 2021-10-26 | 广东唯泰生物科技有限公司 | Induction medium and use method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
| WO2013184809A1 (en) * | 2012-06-05 | 2013-12-12 | The Regents Of The University Of California | Methods and compositions for the rapid production of retinal pigmented epithelial cells from pluripotent cells |
| WO2014121077A2 (en) * | 2013-02-01 | 2014-08-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | METHOD FOR GENERATING RETINAL PIGMENT EPITHELIUM (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCs) |
| WO2015175504A1 (en) * | 2014-05-12 | 2015-11-19 | The Johns Hopkins University | Differentiation of human pluripotent stem cells into retinal pigment epithelium using hif1 inhibitors |
| CN105814192A (en) * | 2013-10-09 | 2016-07-27 | 加利福尼亚大学董事会 | Methods of mammalian retinal stem cell production and applications |
-
2016
- 2016-12-19 CN CN201611181167.8A patent/CN106754718A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
| WO2013184809A1 (en) * | 2012-06-05 | 2013-12-12 | The Regents Of The University Of California | Methods and compositions for the rapid production of retinal pigmented epithelial cells from pluripotent cells |
| WO2014121077A2 (en) * | 2013-02-01 | 2014-08-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | METHOD FOR GENERATING RETINAL PIGMENT EPITHELIUM (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCs) |
| CN105814192A (en) * | 2013-10-09 | 2016-07-27 | 加利福尼亚大学董事会 | Methods of mammalian retinal stem cell production and applications |
| WO2015175504A1 (en) * | 2014-05-12 | 2015-11-19 | The Johns Hopkins University | Differentiation of human pluripotent stem cells into retinal pigment epithelium using hif1 inhibitors |
Non-Patent Citations (3)
| Title |
|---|
| DAVID E. BUCHHOLZ ET AL.: "Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium", 《STEM CELLS TRANSLATIONAL MEDICINE》 * |
| LYNDSAY L. LEACH,ET AL.: "Canonical/b-Catenin Wnt Pathway Activation Improves Retinal Pigmented Epithelium Derivation From Human Embryonic Stem Cells", 《NANOTECHNOLOGY AND REGENERATIVE MEDICINE》 * |
| 高珍 等: "小分子化合物在干细胞诱导分化为视网膜色素上皮细胞中的应用", 《眼科新进展》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906178A (en) * | 2017-04-07 | 2017-06-30 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Cultivate the application in the composition and its holding epithelioid cell's form of epithelial cell |
| CN108531443A (en) * | 2018-04-04 | 2018-09-14 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | The method that small molecule inducing pluripotent stem cells are divided into retinal pigment epithelium |
| CN109136184A (en) * | 2018-07-16 | 2019-01-04 | 同济大学 | The method that induction people's multipotent stem cells are divided into RPE cell |
| CN109136184B (en) * | 2018-07-16 | 2021-09-03 | 同济大学 | Method for inducing differentiation of human pluripotent stem cells into RPE cells |
| CN110628696A (en) * | 2019-08-28 | 2019-12-31 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
| CN110628696B (en) * | 2019-08-28 | 2021-09-07 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
| CN113549596A (en) * | 2021-08-04 | 2021-10-26 | 广东唯泰生物科技有限公司 | Induction medium and use method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754718A (en) | A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium | |
| Brewer | Isolation and culture of adult rat hippocampal neurons | |
| Zhao et al. | Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells | |
| Jin et al. | Generation of retinal cells from pluripotent stem cells | |
| JP2005514926A (en) | Novel mammalian multipotent stem cells and compositions, methods for their preparation and administration | |
| CN105940101A (en) | Specification of functional cranial placode derivatives from human pluripotent stem cells | |
| CN110042082A (en) | Retinal pigment epithelium and its preparation method and application | |
| CN102952777A (en) | Inducing method for directionally differentiating human embryonic stem cells to corneal endothelial cells | |
| US20050196864A1 (en) | Induction and high-yield preparative purification of mesencephalic dopaminergic neuronal progenitor cells and dopaminergic neurons from human embryonic stem cells | |
| Viczian | Advances in retinal stem cell biology | |
| Edamura et al. | Recombinant canine basic fibroblast growth factor-induced differentiation of canine bone marrow mesenchymal stem cells into voltage-and glutamate-responsive neuron-like cells | |
| CN105255836A (en) | Method for preparing person stem cells with improved neural restoration function and application of person stem cells | |
| WO2013071469A1 (en) | Method, kit, and application of applying reverse differentiation to human somatic cells to generate autologous retinal stem cells and autologous retinal cells | |
| KR100734714B1 (en) | Process for producing retinal neurocyte from neural stem cell derived from iris tissue and retinal neurocyte produced by the process | |
| CN110302398A (en) | A kind of composition containing Atoh7 and/or Pou4f, preparation method and medical usage | |
| CN113528441B (en) | Rapid and efficient clinical-grade pigment epithelial cell induction method, kit and application | |
| Pellitteri et al. | Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth | |
| Kang et al. | Application of human stem cell derived retinal organoids in the exploration of the mechanisms of early retinal development | |
| CN109666642B (en) | Method for in vitro separation and purification of oligodendrocyte precursor cells of tree shrew cerebral cortex | |
| CN109456937A (en) | A kind of culture medium preparing Endometrial stem cell and preparation method | |
| WO1997001628A1 (en) | Liquid medium for nerve cells, process for producing the same and method for incubating nerve cells with the use of the same | |
| RU2430158C1 (en) | Method for differentiation of adult human stem cells in insulin-secreting cells | |
| JP2002325571A (en) | Method for inducing differentiation of retina | |
| CN110199985A (en) | A kind of preparation method of neuron frozen stock solution | |
| Luo et al. | Stem cell therapies for glaucoma and optic neuropathy |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
| RJ01 | Rejection of invention patent application after publication |