CN108795864A - A method of obtaining the class retinal tissue rich in the cone and rod cell using people's induced multi-potent stem cell - Google Patents

The invention discloses a kind of methods obtaining the class retinal tissue rich in the cone and rod cell using people's induced multi-potent stem cell.It includes digesting hiPSCs to obtain cell precipitation, then cell precipitation is suspended to cultivate and obtains embryoid body；Embryoid body is inoculated to in the coated culture dishes of Matrigel, the induction differentiation in induction broth obtains neural retina (NR) and RPE in advance；NR and RPE are provoked again, suspend culture, it includes NR tissues to obtain 3D class retinas, then continue the culture that suspends in the optimization culture liquid of not retinoic acid, NR differentiates all retina cells, includes the photoreceptor cell of high mature, Rhodopsin positive rod cells, the positive red green cone cells of L/M OPSIN and the positive blue cone cells of S-OPSIN, especially obtain the class retinal tissue rich in the red green cone and rod cell.

It is a kind of to be regarded using class of people's induced multi-potent stem cell acquisition rich in the cone and rod cell The method of omental organization

Technical field：

The invention belongs to stem cell regenerating biological fields, and in particular to multipotential stem cell regenerates class retina organ, obtains The method of retina seed cell.

Background technology

Retina degenerative disease is the main eye disease for causing irreversibility to be blinded, mainly due to photoreceptor cell, RPE Cell or gangliocyte are by irreversible damage.However, such disease there is no effective therapy at present.Stem-cell therapy New hope of recovering lost eyesight is brought for such disease, acquisition is similar with human retina cell characteristics, and the kind of quantity abundance is careful Born of the same parents are the key that retina degenerative disease is recovered lost eyesight treatment and disease mechanisms research.

Human retina cell was mostly detached from the Embryonic Retina of miscarriage and was obtained in the past, but quantity is very limited, batch matter Amount etc. is difficult to control, and be cannot be satisfied the needs of clinical treatment, is limited the development of retina cell's treatment technology.In recent years, people Retina cell can also include that embryo is dry thin from human pluripotent stem cells (human pluripotent stem cell, hPSC) Born of the same parents (ESC) and induced multi-potent stem cell (iPSC) Induction of committed differentiation, for retina seed cell provide it is new come Source.Currently, there are many induction differentiation schemes, and hPSC to be instructed gradually to break up to retina cell.First, it is adherent (i.e. in classics Two dimension, 2D) under condition of culture, hPSC has not only differentiated a variety of retina cells, including gangliocyte, RPE cells and Photoreceptor cell etc., can also directly induce original optic vesicle spline structure, and the latter further differentiates a variety of retina cells； Second, with the development of 3D suspended tissues Fiber differentiation system, 3D retina method for inducing and cultivating is born.Japanese Scientists profit With the method for suspension EBs, 3D optic vesicles are obtained from the ESC of mouse and people differentiation respectively, the latter further differentiates various developments In retina cell；Third, China scientist professor Zhong Xiufeng use the inducing culturing condition that 2D is combined with 3D, develop A set of retina induces differentiated system, and first hiPSCs suspends and cultivates, and obtains embryoid body EBs, then by EBs again adherent differentiation, The 3D class retinal tissues with layered structure, including all main retina cells are obtained, can be obtained after long-term cultivation Has functional photoreceptor cell.

Although the technology of multipotential stem cell induced retinal makes great progress, current retina induces differentiation scheme It comes with some shortcomings.First, most induction schemes are more complex, need to add various cell factors or signaling molecule, including regard Huang Sour A (retinoic acid, RA)；Second, the retinal light injury photoreceptor obtained is based on rod cell, and cone cell is very It is few.Cone cell is responsible for daylight vision or photopic vision, color and crack vision, concentrates on the macular region of retina.Retinal rod is thin Born of the same parents are responsible for night vision or noctovision, are distributed in the peripheral portion of retina.The mankind mainly in activity on daytime, therefore, depend on The function of cone cell.Age associated retinal maculopathy has become the main diseases causing blindness of 50 years old global or more crowd, main It will be caused by cone cell degeneration death.Therefore, current retina induction scheme is difficult to meet needed for clinical treatment.

Invention content：

For existing retina induction differentiation technique there are the problem of (complicated component, it is difficult to obtain retinal disease treatment Required cone cell), the present invention provides a kind of class of improvement, the simple, effective acquisition rich in retinal rod and cone cell The induction scheme of retina.

The side that the class retinal tissue rich in the cone and rod cell is obtained using people's induced multi-potent stem cell of the present invention Method, including hiPSCs Clone Digestions are obtained into cell precipitation, then cell precipitation is suspended to cultivate and obtains embryoid body；Embryoid body is again It is seeded in advance in the coated culture dishes of Matrigel, differentiation is induced in induction broth, obtains neural retina (neural retina, NR) and RPE；NR and RPE are provoked again, suspend culture, obtain 3D class retinas include NR tissue and RPE.Using the culture solution long-term suspension culture without RA, NR can not only develop all retina cells, including height Ripe photoreceptor cell, Rhodopsin positive rod cells, the positive red green cone cells of L/M OPSIN and S-OPSIN sun Property indigo plant bore cell, be especially that of obtaining NR or class retinal tissue rich in retinal rod and red green cone cell.

First purpose of the present invention provides a kind of method of digestion hiPSCs clones, it is characterised in that preferably, described The cell precipitation that obtains hiPSCs Clone Digestions be to clone hiPSCs to be digested with EDTA to obtain cell precipitation.With dispase Digestion is compared, and EDTA digestion has the following advantages that：1) with EDTA digestion hiPSCs clone can gently blow and beat be dispersed into it is small Piece, and be mostly small agglomerate with the postdigestive products of dispase, so that the size and homogeneity to the formation of later stage EBs cause It influences；2) the hiPSCs cells of identical quantity while with EDTA and dispase are digested, the EBs quantity obtained after EDTA digestion is remote The EBs amounts obtained much larger than dispase digestion；3) EDTA is chemical reagent, non-animal source source, convenient for clinical Transformation Application； And dispase is protease, and it is variant between batch, it is animal sources source, is unfavorable for clinical Transformation Application；4) when EDTA digests Between it is short, at a temperature of digestion at 37 degree, about 3-6min is activity stabilized controllable；And dispase digestions time are long, activity is unstable, At a temperature of digestion at 37 degree, 10-20min, bad grasp condition, poor repeatability are needed.

It is preferred that it is with 0.5 μM of EDTA, 37 DEG C of digestion 5min that the EDTA, which digests,.

Second object of the present invention is efficiently simply differentiated from hiPSCs inductions rich in largely regarding there is provided a kind of The method for boring the 3D retinas of cell and rod cell comprising obtain 3D retinal tissues, then 3D retinal tissues exist It is cultivated in culture solution, which is characterized in that be to carry out 3D retinal tissues in the optimization culture liquid without containing retinoic acid Culture.3D retinal tissues after long-term cultivation develop ripe photoreceptor cell, and there are about 43% 3D class retina groups It knits rich in the positive red green cone cells of L/M OPSIN, is reported far above previous research.

By postdigestive hiPSCs cells, suspension culture is carried out by the way that 10mM Blebbistatin are added, generates embryoid body (EBs), it inoculates to using in the coated culture dishes of Matrigel in advance, carries out retina induction differentiation with culture solution, differentiate Retina and RPE provoked with Tungsten needles and carry out suspension culture, clear layer is obtained after long-term cultivation, rich in ripe light The 3D retinal tissues of receptor cell.According to induction atomization culture medium successively be mTeSR1, NIM culture solution, RDM culture solutions, RC2 culture solutions, RC1 culture solutions, mTeSR1 are that hiPSCs maintains culture solution, NIM culture solutions to be lured for early stage nerve Culture solution is led, RDM culture solutions are retina induction broth, and RC2 culture solutions and RC1 culture solutions are 3D retinal tissue cultures Liquid.The RC2 culture solutions and RC1 culture solutions is RC2/RA culture solutions, the RC1/RA culture solutions for not adding retinoic acid.Do not add On the one hand induction that retinoic acid reduces exogenous molecules is added to influence, on the other hand having saved the cost of culture medium, (every other day plus RA is changed New culture solution loss mass propgation liquid).In addition, retinoic acid is the signal of interest molecule of retinal photoreceptor Development And Differentiation, Previous studies show that the RA of high concentration inhibits reaching maturity for cone cell.The culture of retinoic acid is not added in the present invention Base promotes reaching maturity for photoreceptor cell, has especially induced more red green cone cells, has been expected to as people's view The cell therapy of film maculopathy provides required seed cell.

It is that 3D retinal tissues successively exist that the 3D retinal tissues carry out culture in the culture solution without containing RA It is cultivated in RC2 culture solutions and RC1 culture solutions, the RC2 culture solutions and RC1 culture solutions are referred respectively to without RA (depending on Huang Acid) RC2/RA culture solutions, RC1/RA culture solutions.

Beneficial effects of the present invention are as follows：

With EDTA digest hiPSCs come start differentiation and prepare EB advantage it is as follows：1) with hiPSCs grams of EDTA digestion Grand can gently blow and beat is dispersed into small pieces, and is mostly small agglomerate with the postdigestive products of dispase, so as to later stage EBs shape At size and homogeneity impact；2) the hiPSCs cells of identical quantity while with EDTA and dispase are digested, The EBs quantity obtained after EDTA digestion is far longer than the EBs amounts that dispase digestion obtains；3) EDTA is chemical reagent, non-dynamic Material resource source, convenient for clinical Transformation Application；And dispase is protease, and it is variant between batch, it is animal sources source, is unfavorable for Clinical Transformation Application；4) EDTA digestions time are short, and at a temperature of digestion at 37 degree, about 3-6min is activity stabilized controllable；And Dispase digestions time are long, and activity is unstable, at a temperature of digestion at 37 degree, 10-20min, bad grasp condition needed to repeat Property is poor.

RA is a kind of fat-soluble small molecule, is the metabolite of vitamin A (VitaminA), in embryo development procedure Have great importance.In eye growth course, RA forms optic rudiment, and cell Proliferation and differentiation play an important roll.Except this Except, research in vivo and in vitro has been found that RA has important adjustment effect to the development of photoreceptor cell.Based on above-mentioned reason By to promote photoreceptor cell to reach maturity, with ESC or iPSCs to the induction scheme of photoreceptor differentiation, culture medium All more or less adds RA as derivant, as a result, the photoreceptor cell induced in such systems with Based on rod cell, cone cell is seldom.

The 3D retina Differentiation Systems of the present invention dexterously solve the problems, such as this, not only obtain the class rich in rod cell Retinal tissue also obtains the retinal tissue rich in red green cone cell for the first time.Compared with other induction differentiation schemes, New departure of the present invention does not add small molecule inducer RA, in the culture of the system during retinal tissue long-term cultivation In the process, 3D retinas still physically well develop, and layered structure, early development is kept to go out main gangliocyte and centre god Through member, ripe photoreceptor cell is developed in the later stage, and there are the 3D retinal tissues of about half thin rich in the red green cone Born of the same parents.Innovatively propose new viewpoint, RA is in vitro in retinal light injury photoreceptor maturation process during fetal growth, it is not necessary to add Ingredient, in the induced environment for lacking RA, 3D retinal developments go out the tissue rich in red green cone cell.The advantage of the invention It is 1) to obtain the retinal tissue for being largely rich in cone cell；2) it reduces foreign constituents RA to be added, promotes the clinic of inducible system Transformation Application；3) culture medium cost and labour costs are saved, pollution risk is reduced.In previous research, extremely not due to RA Stablize, need frequent (what is had is daily) supplement and replace culture solution, have lost mass propgation liquid, a large amount of manpowers is needed to remain thin Born of the same parents, and increase the risk of cell contamination.

Description of the drawings：

Fig. 1 is EDTA and digestion initial stages and EB total amount comparison diagram of the dispase to hiPSCs：Scheme the hiPSCs of A equal parts Respectively with the cell precipitation for obtaining similar amount after EDTA (left figure) and dispase (right figure) digestion；Figure B uses EDTA respectively The postdigestive hiPSCs of (left figure) and dispase (right figure) carries out EB preparations, after EB centrifugations in the 6th day, EDTA digestion The EB precipitations (left figure) that hiPSCs is obtained are bigger than the EB precipitations (right figure) that the hiPSCs that EDTA digests is obtained.

Fig. 2 is the influence diagram for comparing EDTA and being formed to EB with dispase digestion process：It is to use respectively to scheme A, figure B and figure C The EB forms and quantity of D0, D3 and D6 after EDTA digestion；It is D0, D3 and D6 after being digested with dispase respectively to scheme D, figure E and figure F EB forms and quantity, EDTA digestion gained EB forms are relatively more uniform, and quantity is more.

Fig. 3, which is no RA optimizations inducible system, does not influence the differentiation of 3D retina early and middle portions：Scheme A and figure B shows 3D views Film (w6) early expression retinal precursor cells marker PAX6 and VSX2, figure C show that early stage 3D retina (w6) develops The gangliocyte of the BRN3 positives, figure D show that mid-term 3D retinas (w13) develop the amakrine of the AP2 positives.

Fig. 4 is the class retinal tissue (after differentiation 34 weeks) of no RA optimizations Fiber differentiation system long-term cultivation.It is low power to scheme A Figure；Figure B is the partial enlarged view of figure A, shows the acromere of hairbrush shape.

Fig. 5 is that immunofluorescence shows that the part class retinal tissue of no RA systematic cultivations is rich in Rhodopsin positive retinal rods Cell (w21).

Fig. 6 is that immunofluorescence shows that the part class retinal tissue of no RA systematic cultivations is rich in red green cone cell：Scheme A Showed by immune group result 3D retinal tissues are rich in the red green cone cell of a large amount of L/M OPSIN positives, and figure B is figure A whites The enlarged drawing of box, the rod cell that the Rhodopsin positives in A same class retinal tissues are schemed in figure C displays are reduced, and figure D is Scheme the enlarged drawing of C white box.

Fig. 7 is that immunofluorescence shows that the class retinal tissue of no RA optimization systems culture contains a small amount of S-OPSIN positives Blue cone cell (w21).

Specific implementation mode：

The following examples are further illustrations of the invention, rather than limiting the invention.

Embodiment 1：

One, the maintenance culture of hiPSCs

1, hiPSCs cells：Mankind's induced multi-potent stem cell in urine source, cell line UE017

2, reagent and consumptive material：

1) mTeSR1 culture mediums：STEM CELL, #05851,4 DEG C

2)EDTA：Invitrogen, 15575-038, room temperature

3) dispase, sigma, D4693, room temperature

4)PBS(1X)：Ji Nuo biological medicine technologies Co., Ltd, 14111202, room temperature

5)Matrigel：Corning, 354277, -20 DEG C

6) six orifice plate：FALCON, 353046

7) centrifuge tube：BD FALCON, 352096

3, instrument

1)CO₂Incubator：SANYO, MCO-20A1C

2) inverted microscope：Nikon, TS100

4, step：

HiPSCs maintenances are incubated in mTeSR1 culture mediums, are passed on when density is grown to about 80%-90% floor spaces (about 4-5 days), are digested with the dispase of the EDTA of 0.5mM or 1mg/mL, and postdigestive cell kind is in Matrigel packets On the culture plate of quilt.

Two, the influence that the digestion method of hiPSCs forms embryoid body (EBs)

1, cell：The hiPSCs cells of step 1 culture

2, reagent and consumptive material：

1) mTeSR1 culture mediums：STEM CELL, #05851,4 DEG C

2) NIM culture mediums (formula is shown in Table 1, and preparation method is to be uniformly mixed each ingredient by its content)

3)EDTA：0.5M EDTA solution, Invitrogen, 15575-038, room temperature

4) dispase, sigma, D4693, room temperature

5)Blebbistatin：sigma

6)Matrigel：Corning, 354277, -20 DEG C

7) low adsorption ware (100mm)：

3, instrument：

1)CO₂Incubator：SANYO, MCO-20A1C

4, step：

EB is prepared with EDTA digestion hiPSCs：It is differentiated thin during the hiPSCs of step 1 is cloned under an optical microscope Born of the same parents strike off, remaining to suck culture solution, are cleaned one time with sterile PBS, and 0.5mM EDTA are added, and 37 DEG C of incubation 5min use pipette tips It gently blows and beats 3-5 time or so, collects cell suspension, 1000rpm, centrifugation 4min, cell precipitation is with containing 10 μM The culture solution of Blebbistatin is resuspended, 37 DEG C of cultures that suspend, overnight.

EB is prepared with dispase digestion hiPSCs：It first under an optical microscope will be differentiated in the hiPSCs of step 1 Cell strikes off, remaining to suck culture solution, is cleaned one time with sterile PBS, and the dispase of 1mg/ml, 37 DEG C of incubations are added 15min is gently blown and beaten 5 times or so with pipette tips, collects cell suspension, and 1000rpm centrifuges 4min, and cell precipitation, which is used, contains 10 μM Blebbistatin culture solution be resuspended, 37 DEG C suspend culture, overnight.

The EDTA or dispase cell precipitations digested are resuspended in the culture solution containing 10 μM of Blebbistatin respectively In, it is transferred to low adsorption ware, 37 DEG C of overnight incubations, EBs prepares the same day and is labeled as the 0th day, and next day is denoted as the 1st day, the 1st day, Culture solution replaces with volume fraction 25%NIM (formula is shown in Table 1)+volume fraction 75%mTeSR1；2nd day, culture solution replaced with Volume fraction 50%NIM+ volume fraction 50%mTeSR1 all change NIM culture solution cultures into since third day, continuous outstanding Floating culture was to the 6-7 days.Visible EBs is formed within second day, continuous to keep culture 6-7 days that suspends, and is centrifuged respectively, and observation precipitation is big Small (Fig. 1).

As shown in Figure 1, the hiPSCs of identical quantity uses EDTA or dispase to digest respectively, Fig. 1-A show two kinds of sides The postdigestive cell precipitation of method is almost the same；Fig. 1-B displays, the EB precipitations after suspending 6 days, the EBs amounts that EDTA digestion obtains are bright It is aobvious to be digested higher than dispse.

Fig. 2 is the influence for comparing EDTA and being formed to EB with dispase digestion process, as shown in Fig. 2, identical quantity HiPSCs uses EDTA or dispase to digest respectively, and Fig. 2-A and Fig. 2-D show the thin of the EDTA and dispase digestion same day respectively Born of the same parents' agglomerate size and number, the postdigestive cells of EDTA relatively disperse it is uniform, and the digestion product of dispase some be in strip, Same day observation cell quantity does not have notable difference；Fig. 2-B and Fig. 2-E are respectively the EBs after assembling 3 days, and EDTA digestion obtains EBs quantity is significantly more than dispase；Fig. 2-C and Fig. 2-F are respectively the EBs after 6 days, and it is apparent that EDTA digestion obtains EBs quantity More than dispase.

Three, 3D retinas break up and are formulated induction photoreceptor maturation without RA

1, cell：hiPSCs(UE017)

2, reagent and consumptive material：

1) RDM culture solutions：Formula is shown in Table 2, and preparation method is to be uniformly mixed each ingredient by its content；

2) RC2 culture solutions：Formula is shown in Table 3, and preparation method is to be uniformly mixed each ingredient by its content；

3)RC1^-Culture solution：Formula is shown in Table 4, and preparation method is to be uniformly mixed each ingredient by its content；

4) 24 orifice plate of low adsorption or low adsorption ware：BIOFIL, 170313-076

3, instrument：

1)CO₂Incubator：SANYO, MCO-20A1C

4, step：

HiPSCs is digested with above-mentioned EDTA schemes, and the culture that suspends obtains EBs.The digestion same day is labeled as the 0th day (D0), Next day is denoted as the 1st day, and so on.6th or 7 day, by EBs renewed vaccinations in using the coated culture dishes of Matrigel in advance, Start adhere-wall culture induction differentiation.Breaking up 4th week, the regions NR and RPE preliminarily form, can be recognized under inverted microscope, and It is provoked in tungsten filament, is moved into 24 orifice plates or low adsorption ware of low adsorption the culture that suspends, changes liquid weekly 2-3 times.Suspend culture Under the conditions of, NR developments thicken, and gradually form translucent ring-type, the subsidiary more or less RPE in one end, referred to as " 3D class retinas Tissue ", and can continue to break up with long-term cultivation, NR, Development And Differentiation goes out all retinal neuronal cells, including photoreceptor Cell.Culture solution scheme (bibliography Xiufeng Zhong, Nature Communications similar with having published an article 2014), in addition to being not added with any RA, specially：The 6-15 days, nerve-inducing is carried out using NIM (table 1), is used within the 16-41 days RDM culture solutions (table 2), from the 42-90 days, 3D retinas were cultivated with the RC2 culture solutions (table 3) that FBS is added, from the 91st It starts to carry out longer-term culture with RC1 culture solutions (table 4).

RA is typically considered to have the function of that retinal development and photoreceptor cell are ripe, former scheme (existing skill Scheme in art, table 5RC2/RA culture solutions and table 6RC1/RA culture solutions, bibliography Xiufeng Zhong, Nature Communications 2014) in addition RA induced, from the 42-90 days or the 63-90 days, with containing 1 μM of RA RC2 culture solutions were daily or change liquid (table 5, preparation method are to be uniformly mixed each ingredient by its content) every other day, from 91 days More than, changing the RC1 culture solutions containing 0.5 μM of RA every other day, (table 6, preparation method are to be uniformly mixed each ingredient by its content ), the differentiation of induction 3D retinas and photoreceptor are ripe.

And in the present invention, 3D retinal tissues still select to use RC2 and RC1 culture solutions respectively at above-mentioned two time point Culture, but RA inductions are added without, the formula of specific culture solution is as shown in Table 3 and Table 4.Every batch of chooses 3-5 3D retina of early stage Tissue, it is fixed, it is dehydrated, slice carries out immunofluorescence dyeing identification.As a result illustrate, the 3D retinas in the inducible system of no RA Development is not affected by influence, and equally follows the development course of retina.From figure 3, it can be seen that in the solution of the present invention, HiPSCs equally follows the development course of retina, the 3D view film expression early stage neural retina markers at the initial stage that suspends PAX6 and VSX2 (Fig. 3-A and 3-B), development mid-term generate BRN3 positive gangliocyte (Fig. 3-C) and AP2 the positive Amakrine (Fig. 3-D).

By being up to the culture of 17 weeks or more (longest 34 weeks), it is thin that 3D retinal tissues differentiate ripe photoreceptor Born of the same parents.As can be seen that Fig. 4-A are in RC2 and RC1 culture mediums in Fig. 4, the 3D retinas of long-term cultivation are carried out, about 34w is regarded Nethike embrane；Fig. 4-B show that the retinal tissue contains a large amount of outer section structures.The 3D retinal tissues of long-term cultivation, every batch of choose 3- It is 5, fixed, it is dehydrated, slice carries out immunofluorescence dyeing identification (general technology).As a result, it has been found that Differentiation System production of the present invention About 50% 3D retinal tissues are (7/14, n=based on the rod cell of the Rhodopsin positives in raw retinal tissue 14), Fig. 5 is shown in the optimization culture environment of no RA additions, and the retinal rod that 3D retinas contain a large amount of Rhodopsin positives is thin Born of the same parents.Importantly, there are about the red green cone cells (6/ that 43% 3D retinal tissues contain a large amount of L/M OPSIN positives 14, n=14), Fig. 6-A add for no RA in environment, and 3D retinas contain the cone cell of a large amount of L/M OPSIN positives, Fig. 6- B is the enlarged drawing in the white box of left side, and Fig. 6-C show that the rod cell ratio of the Rhodopsin positives in the tissue is reduced, Fig. 6-D are the enlarged drawing in the white box of left side.Fig. 7 is shown in the culture environment of no RA additions, and 3D retinas contain a small amount of S The blue cones cell of the opsin positives.And the 3D retinal tissues that prior art systems obtain, 90% or more is Rhodopsin sun Property rod cell, the red green cone cell of a small amount of L/M OPSIN positives and a small amount of S-OPSIN positive blue cone cell.Therefore, Compared with prior art, present system can obtain the view of the red green cone cell more containing the L/M OPSIN positives Membrane tissue, and the blue cones cell proportion of the S-OPSIN positives is still smaller, it is almost the same with research before.

Table 1NIM culture solutions (the 3-15 days)

Table 2RDM culture solutions (the 16-41 days)

Table 3RC2 culture solutions (the 42-90 days) (present invention)

Table 4RC1 culture solutions (after the 91st day) (present invention)

Table 5RC2/RA culture solutions (the 42-90 days) (old scheme)

Table 6RC1/RA culture solutions (after the 91st day) (old scheme)