CN101052305B

CN101052305B - Isolated lineage negative hematopoietic stem cells and methods of treatment therewith

Info

Publication number: CN101052305B
Application number: CN2005800379820A
Authority: CN
Inventors: M·弗里德兰德; 大谷笃史; K·达西尔瓦; S·(H·)莫尔诺
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2004-09-03
Filing date: 2005-09-02
Publication date: 2012-10-10
Anticipated expiration: 2025-09-02
Also published as: KR101309500B1; EP1802199A2; CA2579292A1; AU2005285246B2; CN101052305A; KR20070104512A; MX2007002639A; AU2005285246A1; JP2008512376A; EP1802199A4; ZA200702607B

Abstract

Isolated, mammalian, adult bone marrow-derived, lineage negative hematopoietic stem cell populations (Lin<-> HSCs) contain endothelial progenitor cells (EPCs) capable of rescuing retinal blood vessels and neuronal networks in the eye. Preferably at least about 20% of the cells in the isolated Lin<-> HSCs express the cell surface antigen CD31. The isolated Lin<-> HSC populations are useful for treatment of ocular vascular diseases. In a preferred embodiment, the Lin<-> HSCs are isolated by extracting bone marrow from an adult mammal; separating a plurality of monocytes from the bone marrow; labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens; removing of monocytes that are positive for the lineage surface antigens from the plurality of monocytes, and recovering a Lin<-> HSC population containing EPCs. Isolated Lin<-> HSCs that have been transfected with therapeutically useful genes are also provided, and are useful for delivering genes to the eye for cell-based gene therapy.

Description

Isolating lineage negative hematopoietic stem cells and with the method for its treatment

The cross reference of related application

The application is the u.s. patent application serial number of submitting on April 28th, 2,004 10/833; 743 part continuation application, u.s. patent application serial number 10/833,743 are u.s. patent application serial numbers 10/628 of submitting on July 25th, 2003; 783 part continuation application; U.s. patent application serial number 10/628,783 requires temporary patent application sequence number of submitting on July 25th, 2,002 60/398,522 and the temporary patent application sequence number of submitting on May 2nd, 2,003 60/467; 051 rights and interests, whole disclosures of above-mentioned application all are attached among this paper by reference.

Statement of government interest

Part of work described herein is subsidized by American National ICR subsidy CA92577 and the subsidy EY11254 of NIH, EY12598 and EY125998.United States Government has some right in the present invention.

Invention field

The present invention relates to isolating mammalian stem cell.More particularly, the present invention relates to the lineage negative hematopoietic stem cells (Lin of bone marrow ^-HSC) crowd, and through using isolating Lin ^-HSC crowd treats the method that mammals eyes keeps suffering from the cone cell in the Mammals retina of a degenerative disorders.

Background of invention

AMD (ARMD) is the main cause of visual loss in industrialized country with diabetic retinopathy (DR), due to unusual retinal vessel new life.Because retina is made up of well-defined neurone layer, neuroglia layer and blood vessel element layer, so can cause sight function significantly to be lost such as the disorder that visible is relatively little in blood vessel hyperplasia or oedema.Hereditary retinal dystrophy, retinitis pigmentosa (RP) for example, also the aberrant angiogenesis with and vascular atrophy narrow such as arteriole is relevant.The retinal rod sight sensor is attacked in the sex change of most of heredity human retina clearly, but also has the cone forfeiture of following, and the cone is the main cell element of macula lutea, and macula lutea is the zone of being responsible for concentrated, meticulous visual acuity in the human retina.Cone specificity survival factors is existing recently to be described (Mohand-Said etc., 1998, Proc.Natl.Acad.Sci.USA 95:8357-8362), can be beneficial to the cone survival in the retinal degeneration mouse model.

The individuality of hereditary retinal dystrophy influence many 1/3500; It is characterized in that carrying out property yctalopia, defect of visual field, optic atrophy, arteriole attenuate, blood vessel sexually revises thoroughly and can develop into the total blindness's damaged (Heckenlively in center, the visual field usually; J.R. compile 1988; RetinitisPigmentosa, Philadelphia:JB Lippincott Co.).The molecular genetics analysis of these diseases identifies sudden change in more than 110 kinds of different genes, only less relatively percentile invasion and attack individual (Humphries etc., 1992, the Science 256:804-808 of receiving explained in these sudden changes; Farrar etc., 2002, EMBO is J.21:857-864).Have in these sudden changes a lot of all with the light transducer that comprises Visual purple, cGMP phosphodiesterase, rds peripherin and RPE65 in enzyme and structural constituent relevant.Although these observationss are arranged, but still do not slow down or reverse effective therapy of these retinal degenerative diseases progress.When the sight sensor of the animal that the wild-type transgenic is delivered to specific sudden change or retinal pigment epithelium (RPE); The new development of gene therapy realized rds in the mouse (Ali etc., 2000, Nat.Genet.25:306-310) and rd (Takahashi etc.; 1999; J.Virol.73:7812-7816) RPE65 phenotype (Acland etc., 2001, successful reverse Nat.Genet.28:92-95) in phenotype and the dog.

Knownly for many years in the normal adult recycle system and marrow, there is a population of stem cells.The different subgroups of these cells can be according to the positive (Lin of hematopoietic lineage ⁺) or lineage negative (Lin ^-) the pedigree differentiation.In addition, shown recently lineage negative hematopoietic stem cells (HSC) crowd contain the endothelial progenitor cells (EPC) that can form blood vessel in vitro and in vivo (referring to Asahara etc., 1997, Science275:964-7).These cells can participate in the blood vessel generation of normal and pathologic birth back (referring to Lyden etc., 2001 Nat.Med.7,1194-201; Kalka etc., 2000, Proc.Natl.Acad.Sci.U.S.A.97:3422-7; And Kocher etc., 2001, Nat.Med.7:430-6); And be divided into multiple non-endothelial cell types, comprise that liver cell is (referring to Lagasse etc., 2000; Nat.Med.6:1229-34), microglia (referring to Priller etc., 2002 Nat.Med.7:1356-61), myocardial cell are (referring to Orlic etc., 2001; Proc.Natl.Acad.Sci.U.S.A.98:10344-9) and epithelial cell (referring to Lyden etc., 2001, Nat.Med.7:1194-1201).Although these cells have been used for the experimental model that some blood vessels take place, the mechanism of EPC target new vessel is still unknown, and does not still confirm effectively to increase the strategy to the useful cell quantity of particular blood vessel system.

The hemopoietic stem cell that derives from marrow is present unique stem cell type that generally is used for therepic use.Marrow HSC is applied to transplant above 40 years.At present, studying the advanced method of the stem cell of results purifying, to develop treatment white blood disease, lymphoma and the hemopathic therapy of heredity.In the minority human patients to stem cell philtrum treatment mellitus and late period kidney clinical application study.

Summary of the invention

The present invention provides the method for the cone cell sex change in the Mammals retina that a kind of improvement suffers from eye illness.This method comprises the step that gives hemopoietic stem cell crowd Mammals retina Mammals marrow source, isolating, lineage negative, and said hemopoietic stem cell crowd comprises hemopoietic stem cell and endothelial progenitor cells.Said cell gives with the amount that is enough to postpone the cone cell sex change in the retina.

A kind of preferred method comprises the lineage negative hematopoietic stem cells crowd who is contained endothelial progenitor cells by the Mammals marrow separation of suffering from eye illness, subsequently isolating stem cell is gone into the Mammals intraocular with the amount intravitreal injection that is enough to improve the cone cell sex change in the retina.

The inventive method use derives from hemopoietic stem cell (Lin Mammals marrow, isolating, mammiferous, lineage negative ^-HSC) crowd's (promptly on its cell surface, not expressing the hemopoietic stem cell (HSC) of pedigree surface antigen (Lin)).Preferably, said cell is autologous stem cells (promptly derives from and wait to treat individual mammiferous marrow).Isolating Mammals Lin ^-HSC crowd comprises endothelial progenitor cells (EPC), is also referred to as endothelial precursor cell, its retina astroglia cell of targeted activation optionally in intravitreal injection is pleasing to the eye the time.Preferred said Mammals is behaved.

In a preferred embodiment, Lin of the present invention ^-HSC crowd separates as follows: extract marrow by the Mammals that suffers from eye illness; From marrow, separate multiple monocyte; With pedigree colony antibody labeling monocyte to one or more antigenic biotin-conjugated in pedigree surface, remove the monocyte of pedigree surface antigen positive, reclaim the Lin that contains EPC then ^-HSC crowd.Preferably; Monocyte comes mark with the pedigree colony antibody to one or more antigenic biotin-conjugated in pedigree surface, and said pedigree surface antigen is selected from: CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR and CD235a (glycophorin A).Preferably, the isolating Lin of the present invention ^-Among the HSC crowd at least about 20% cell expressing surface antigen CD31.Give Mammals ill eye isolated cells then, preferably give through intraocular injection.In a preferred embodiment, at least about 50% isolating Lin ^-HSC expresses surface antigen CD31, the isolating Lin at least about 50% ^-HSC expresses surface antigen CD117 (c-kit).

Lin of the present invention ^-EPC among the HSC crowd infiltrates in the developmental retinal vessel widely and infiltrates in the amphiblestroid neural network, and still stably infiltrates in eye new vessel and the neural network.The normal mouse retina is mainly retinal rod, still, in the mouse of the inventive method treatment, Lin ^-The cell that HSC treatment back is saved almost all is the cone surprisingly.

In a preferred embodiment, with a kind of isolating Lin of useful gene transfection that treats ^-HSC crowd's cell.For example; Said cell can be used the polynucleotide transfection of efficient coding neurotrophy material or angiogenesis inhibitor material; Said cell selective target new vessel system, and under the situation that does not influence existing blood vessel, suppress neovascularization through a kind of gene therapy form based on cell.In one embodiment, the isolating Lin that is used for the inventive method ^-HSC crowd is contained a kind of gene of the blood vessel generation inhibiting peptide of encoding.Blood vessel generation inhibition Lin ^-HSC is used in such as dysregulation angiogenic growth in the disease of ARMD, DR and some retinal degeneration relevant with the abnormal vascular system.In a further preferred embodiment, isolating Lin of the present invention ^-HSC contains the gene of the neurotrophic peptide of encoding.Neurotrophic Lin ^-HSC can be used for promoting the neurone rescue in the eye illness that said eye illness comprises retina nerve degeneration, for example glaucoma, retinitis pigmentosa etc.

With isolating Lin of the present invention ^-The special advantage that HSC crowd treats eye is to use Lin ^-Observe vascular nutrition property and neurotrophic rescue effect in the eye of handling in the HSC vitreum.With the isolating Lin of the present invention ^-In the eye of HSC treatment, retinal neurons and sight sensor (the especially cone) are able to keep, and some of sight function are measured and can be kept.

Preferably, the ill retina that will treat of the inventive method comprises the activatory astroglia cell.This can realize through early treatment eye when relevant gliosis is arranged, or realize through the local multiplication that uses laser stimulation activatory astroglia cell.

The accompanying drawing summary

In the accompanying drawings:

Fig. 1 illustrates the amphiblestroid synoptic diagram of developmental mouse.(a) growth of elementary clump.(b) subordinate phase of retinal vessel formation.GCL, ganglion-cell layer; IPL, interior clump layer; INL: inner nuclear layer; OPL, outer clump layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; ON, optic nerve; P, periphery.Figure (c) illustrates bone marrow Lin ⁺HSC and Lin ^-The flow cytometry characteristic of HSC isolated cell.The top row: the dot chart of non-antibody labeled cell distributes, and wherein R1 confirms the painted quantitative gate of positive PE-district, and R2 representes that GFP-is positive; Middle line: Lin ^-HSC (C57B/6); End row: Lin ⁺HSC (C57B/6) cell, each clone all uses the PE-of Sca-1, c-kit, Flk-1/KDR, CD31 to put together antibody labeling.The Tie-2 data derive from the Tie-2-GFP mouse.Percentage ratio is represented total Lin ^-HSC or Lin ⁺The per-cent of positive labeled cell among the HSC crowd.

Fig. 2 illustrates Lin ^-HSC implants in the developmental mouse retina.(a) when injection back 4 days (P6), the eGFP of intravitreal injection ⁺Lin ^-The HSC cell adheres on retina and breaks up.(b) Lin ^-HSC (the B6.129S7-Gtrosa26 mouse is with β-gal antibody staining) is setting up himself with the vascular system (the vascular system top represented in asterisk) of collagen protein IV antibody staining is preceding.(c) when injection back 4 days (P6), most of Lin ⁺HSC cell (eGFP ⁺) can not break up.Mesentery eGFP when (d) injecting back 4 days (P6) ⁺Mouse EC.(e) be injected into Lin in the little rathole of adult ^-HSC (eGFP ⁺).(f) eGFP that in the GFAP-GFP transgenic mice, goes back to the nest and break up according to already present star spongiocyte template ⁺Lin ^-The low power of HSC (arrow place) is amplified.(g) Lin ^-Associating high power between cell (eGFP) and the star spongiocyte below it (arrow place) is amplified.(h) the not GFAP-GFP transgenic of injection contrast.When (i) injecting back 4 days (P6), eGFP ⁺Lin ^-HSC is to the district's migration of dark clump and the experience differentiation in future.Left figure has obtained the Lin in the self-contained retina ^-HSC is active; Right figure indication Lin ^-The position of cell (arrow place) in retina (top is a glass dignity, and the bottom is a scleral surface).(J) carry out double-tagging with α-CD31-PE and α-GFP-alexa 488 antibody.Injected the Lin of injection back 7 days ^-HSC (eGFP, redness) infiltrates in the vascular system (CD31).The district is infiltrated in the arrow indication.(k) back 14 days (P17) eGFP of injection ⁺Lin ^-The HSC cell forms blood vessel.The blood vessel that (l and m) intracardiac injection rhodamine-VISOSE is illustrated in elementary clump (l) and the dark clump (m) all is that complete sum has function.

Fig. 3 shows, eGFP ⁺Lin ^-HSC cell-targeting gliosis position (by the star spongiocyte indication of expressing GFAP, the most left figure), it is induced by laser (a) and machinery (b) the two damage of in ripe retina, bringing out (asterisk shows damage location).The rightest figure is an a panel height times enlarged view, shows Lin ^-HSC and star spongiocyte closely associate.Calibration bars=20 μ M.

Fig. 4 shows, Lin ^-The blood vessel of HSC cellular rescue retinal degeneration mouse.(a-d), the retina of the painted injection of collagen protein IV back 27 days (P33); (a) with (b), use Lin ⁺The retina of HSC cell (Balb/c) injection does not show difference with normal FVB mouse aspect vascular system; (c) with (d), use Lin ^-The retina of HSC (Balb/c) injection demonstrates with wild-type mice and similarly enriches blood vessel network; (a) with (c), with the painted whole amphiblestroid frozen section of DAPI (top is that glass is honorable, and the bottom is a scleral surface); (b) with (d), the dark clump of retina total amount; (e), bar graph is illustrated in injection Lin ^-The blood vessel state of the dark plexus vasculosus that forms in the retina of HSC cell increases (n=6).The degree of dark retinal vesselization is come quantitatively through the length overall of calculating every width of cloth image medium vessels.Compared Lin ^-HSC, Lin ⁺HSC or contrast retina medium vessels average total length/high power field (is unit with the micron).(f) with the Lin that derives from the rd/rd mouse ^-HSC cell (R, right eye) or Lin ⁺Carry out the comparison of dark plexus vasculosus length after HSC cell (L, the left eye) injection.The result's (a kind of color is represented a mouse) who has shown 6 independent mouse.(g) with (h) Lin ^-HSC cell (Balb/c) has also been saved the rd/rd vascular system when injecting the P15 eye.Shown injection Lin ^-HSC cell (G) or Lin ⁺The middle plexus vasculosus and the dark plexus vasculosus of the retina of HSC cell (H) (injecting back month).

Fig. 5 illustrates the Photomicrograph of mouse retinal tissue: (a), and the deep layer of retina self-contained (rd/rd mouse), eGFP ⁺Lin ^-HSC injects back five days (P11) visible (grey).(b) with (c), accept Balb/c Lin in P6 ^-Cell (b) or Lin ⁺The P60 retinal blood guard system of Tie-2-GFP (rd/rd) mouse of HSC cell (c) injection.Only visible endogenous endotheliocyte (GFP-dyeing) in (b) and left figure (c).(b) and middle figure (c) use the CD31 antibody staining; Arrow indication with CD31 without the painted blood vessel of GFP, (b) and right figure (c) shown with GFP and CD31 and dyeed simultaneously.(d) injected Lin ^-The retina of HSC (left figure) dyes with the α-SMA of contrast retina (right figure).

Fig. 6 shows, the Lin of T2-TrpRS-transfection ^-HSC suppresses the growth of mouse retinal blood guard system.(a) people TrpRS, T2-TrpRS and have the synoptic diagram of the T2-TrpRS of Igk signal sequence at N-terminal.(b) Lin of injection T2-TrpRS transfection ^-The retina of HSC is expressed T2-TrpRS albumen in vivo.(1) the reorganization T2-TrpRS that in intestinal bacteria, produces; (2) the reorganization T2-TrpRS that in intestinal bacteria, produces; (3) the reorganization T2-TrpRS that in intestinal bacteria, produces; (4) contrast retina; (5) injected Lin ^-The retina of HSC+pSecTag2A (just carrier); (6) injected Lin ^-The retina of HSC+pKLe135 (Igk-T2-TrpRS among the pSecTag).(a), endogenous TrpRS.(b), reorganization T2-TrpRS.(c), injected Lin ^-The retina of the T2-TrpRS of HSC.(c-f), injected back 7 days, accept amphiblestroid representativeness elementary (top layer) clump and secondary (deeply) clump of injection; (c) with (d), injected the Lin of empty plasmid transfection ^-The eye of HSC is grown normal; (e) with (f), the Lin of T2-TrpRS-transfection ^-The eye majority that HSC injected shows dark clump restraining effect; (c) with (e), elementary (surface) clump; (d) with (f), secondary (deeply) clump.Observed weak vessel profile is " seepage " image of the elementary network blood vessel of demonstration in (e) in (f).

Fig. 7 has shown coding His ₆The dna sequence dna SEQ ID NO:1 of the T2-TrpRS of-mark.

Fig. 8 has shown His ₆The aminoacid sequence SEQ ID NO:2 of the T2-TrpRS of-mark.

Fig. 9 illustrates amphiblestroid Photomicrograph of mouse and electroretinogram ERG (ERG), and the eyes of said mouse have been injected Lin of the present invention ^-HSC and Lin ⁺HSC (contrast).

Figure 10 illustrates statistical graph, the clear Lin that uses of this chart ^-The neurone rescue (y-axle) of the middle vascular lamina (Int.) of the rd/rd mouse eyes that HSC handles and dark vascular lamina and the dependency between the blood vessel rescue (x-axle).

Figure 11 illustrates statistical graph, and this figure shows and uses Lin ⁺There is not dependency between the neurone rescue (y-axle) of the rd/rd mouse eyes that HSC handles and the blood vessel rescue (x-axle).

Figure 12 uses Lin ^-The bar graph of the length of vessel (Y-axle) that rd/rd mouse eyes (filaments of sun) that HSC handles and untreated rd/rd mouse eyes (bright wisp) are represented with any relative unit at the time point of back 1 month (1M) of injection, 2 months (2M) and 6 months (6M).

Figure 13 comprises 3 bar graphs, and the nuclear volume of the rd/rd outside nervous layer of mouse (ONR) showed and uses Lin when they were back 1 month (1M) of injection, 2 months (2M) and 6 months (6M) ^-The check figure order of the eye (filaments of sun) that HSC handles is with respect to using Lin ⁺The contrast eye (bright wisp) that HSC handles has remarkable increase.

Figure 14 has described the figure of the outside nervous layer nuclear volume of individual rd/rd mouse, and this figure has compared when the time point of (injection back) 1 month (1M), 2 months (2M) and 6 months (6M) right eye, and (R uses Lin ^-HSC handles) (L uses Lin with left eye ⁺The contrast eye that HSC handles); Every line among the given figure is the right and left eyes of more individual mouse all.

Figure 15 has described the variation of rd1/rd1 mouse (C3H/HeJ, left figure) or middle retinal vessel of wild-type mice (C57BL/6, right figure) and neurocyte.Shown self-contained retina (redness: collagen protein IV, green: CD31) and the middle plexus vasculosus (last figure) in the same retina section (redness: DAPI, green: CD31, figure below) or the retinal blood guard system of deep layer (middle figure) plexus vasculosus.(P: go out DAB).(GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer).

Figure 16 shows, Lin ^-Neurocyte sex change in the HSC injection rescue rd1/rd1 mouse.(A, B and C) injected Lin ^-The injection of the eye of HSC (right figure) and offside control cells (CD31 ^-) the intermediate plexus (int.) of eye (left figure) when P30 (A), P60 (B) and P180 (C) or retinal blood guard system and the section of dark clump, (D), P30 (left figure, n=10), P60 (middle figure, n=10) and P180 (right figure injected Lin in the time of n=6) ^-HSC or control cells (CD31 ^-) amphiblestroid vascular system average total length (MV+or-standard deviation).Data (the Y axle: the relative length of vascular system) that has shown middle plexus vasculosus (Int.) and dark plexus vasculosus respectively.(E), P30 (left figure, n=10), P60 (middle figure, n=10) or P180 (right figure injected control cells (CD31 in the time of n=6) ^-) or Lin ^-Nucleus mean number among the amphiblestroid ONL of HSC (Y axle: nuclear relative number among the ONL).(F), when P30 (left figure), P60 (middle figure) and P180 (right figure), injected Lin ^-HSC or control cells (CD31 ^-) amphiblestroid vascular system length (X axle) and ONL in linear dependence between the cell check figure (Y axle).

Figure 17 shows through injection Lin ^-HSC saves retinal function.Use electroretinogram ERG (ERG) record to measure and injected Lin ^-HSC or control cells (CD31 ^-) amphiblestroid function.(A and B) injects back 2 months by rescue and the amphiblestroid exemplary of not saved.Shown same animal injection Lin ^-The right eye of HSC (A) with injected CD31 ^-The retina section (the painted vascular system of green: CD31, the painted nuclear of redness: DAPI) of the left eye of control cells (B).(C), the ERG result of the same animal shown in (A and B).

Figure 18 shows that human bone marrow cell's group energy is saved sex change retina (A-C) in the rd1 mouse.In another retinal degeneration model rd10, also observe this rescue (D-K).A is with the people Lin of green colouring material mark ^-HSC (hLin ^-HSC) can, intravitreal injection be divided into the retinal blood tube cell after going into C3SnSmn.CB17-Prkdc SCID mouse.(B and C) when injecting back 1.5 months, injecting Lin ^-Retinal blood guard system (left figure in the contrast eye (C) of the eye of HSC (B) or offside; On: intermediate plexus, down: dark clump) with neurocyte (right figure).(D-K), use Lin ^-HSC saves rd10 mouse (when P6, injecting).Shown (D:Lin at P21 ^-HSC, control cells), P30 (E:Lin H: ^-HSC, control cells), P60 (F:Lin I: ^-HSC, control cells) and P105 (G:Lin J: ^-HSC, K: the representative retina in the time of control cells) (the processing eye of each time point and contrast eye are from same animal).(the last figure of every width of cloth figure is an intermediate plexus to the retinal blood guard system; The middle figure of every width of cloth figure is dark clump) with CD31 (green) and collagen protein IV (redness) dyeing.Figure below of every width of cloth figure has shown square section (redness: DAPI, the green: CD31) by same retina preparation.

Figure 19 shows, is using Lin ^-After HSC handled, crystallin α A was raised in the outer nuclear layer cell of rescue, and handle with control cells to branch hole in do not have this phenomenon.Left figure: by the contrast of the IgG in the retina of saving, middle figure: by the crystallin α A in the retina of saving, right figure: the crystallin α A in the retina of not saved.

Figure 20 is included in and uses Lin of the present invention ^-In the muroid retina that HSC handles by the table of up-regulated gene.(A) it is expressed in and uses mouse Lin ^-Increase by 3 times gene in the mouse retina that HSC handles.(B) using mouse Lin ^-The crystal protein gene that is raised in the mouse retina that HSC handles.(C) it is expressed in personnel selection Lin ^-Increase by 2 times gene in the mouse retina that HSC handles.(D) it is expressed in personnel selection Lin ^-The neurotrophic factor that is raised in the mouse retina that HSC handles or the gene of growth factor.

Figure 21 illustrates CD31 and beta 2 integrin alpha 6 surface antigens at the positive (DC133 of CD133 of the present invention ⁺) and the negative (CD133 of CD133 ^-) distribution on the people Lin-HSC crowd.Left figure has shown the flow cytometry dot chart.The figure of central authorities is a histogram with right figure, has shown the expression level of specific antibody on the cell mass.The Y axle is represented event number, and the X axle shows strength of signal.Move to the fluorescent signal and the above antibody expression of background level of the solid histogram representative increase on hollow (contrast) histogram right side.

Figure 22 diagram postnatal P0 day to P30 day retinal development after the birth of the wild-type C57/B16 mouse that normal oxygen level (oxygen level is normal) is raised.

Figure 23 diagram (oxygen level is high with the hyperoxia level during the P7 to P12; 75% oxygen) raise, subsequently by the oxygen induction type retinopathy varying model of P12 to P17 in the C57/B16 mouse that normal oxygen level is raised.

Figure 24 has shown in oxygen induction type retinopathy varying model through using Lin of the present invention ^-HSC crowd handles the blood vessel rescue of carrying out.

Figure 25 shows, intravitreal injection Lin ^-The sight sensor of being saved behind the HSC in the rd1 mouse outer nuclear layer (ONL) is mainly cone cell.The sight sensor of small proportion in the wild-type mice retina (last figure) is a cone cell, and evidence is to express red/green cone opsin (A), and most of cell of ONL is positive to retinal rod specificity Visual purple (B) simultaneously.The retinal blood guard system fluoresces (C) with pre-immune serum self, but stratum nucleare is to negative fully with retinal rod or the dyeing of cone specificity opsin.Rd/rd mouse retina (figure below) has the inner nuclear layer of minimizing and near the complete ONL of atrophy, this two all negative to the cone (D) or retinal rod (figure G) opsin.Contrast CD31 ^-The eye that HSC handles is identical with the rd/rd retina of not injection, and the cone (E) or retinal rod (H) opsin are not had any dyeing.Lin ^-What HSC handled shows obvious reduction but the ONL that clearly exists to branch hole, and it mainly is made up of cone cell, evidence be to the cone red/the positive immunoreactivity (F) of green opsin.Also observe a small amount of retinal rod (I).

Description of Preferred Embodiments

Stem cell can be discerned usually that (detailed discussion is referring to Stem Cells:Scientific Progress and Future Directions by the distribution of antigen on cell surface; The report that science policy office of NIH formulated June calendar year 2001; Appendix E: stem cell labeling, this report is introduced this paper as a reference according to degree of correlation).

The present invention provides the method for the cone cell sex change in the Mammals retina that a kind of improvement suffers from eye illness.Preferably through intravitreal injection will contain hemopoietic stem cell and endothelial progenitor cells Mammals marrow source, hemopoietic stem cell crowd isolating, lineage negative gives the Mammals retina.Said cell gives with the amount that is enough to improve the cone cell sex change in the retina.

A kind of preferred method comprises that by Mammals marrow discrete spectrum to be treated be the negative hematopoietic stem cells crowd, gives Mammals with said cell with the amount that is enough to improve the cone cell sex change in the retina then.

Said cell can derive from ill Mammals, preferably obtains in early days in eye illness.Perhaps, said cell can obtain in premorbid in the known patient who eye illness (for example retinitis pigmentosa) is had a genetic predisposition.Said cell can be stored to when needing, and can freeze to disease in first observed then and prophylactically inject in 1000000 o'clock.Preferred ill retina comprises its activation astroglia cell of stem cell target.Therefore, it is useful when having relevant gliosis, eye being carried out early treatment.Perhaps, available laser therapy retina to stimulate the local multiplication of activation astroglia cell in the retina, gives autologous stem cells then.

Hemopoietic stem cell is the stem cell that can develop into multiple hemocyte type (for example B cell, T cell, granulocyte, thrombocyte and red corpuscle).It is the cell surface protein of mature blood cell pedigree affinity tag that the pedigree surface antigen is one group, comprises CD2, CD3, CD11, CD11a, Mac-1 (CD11b:CD18), CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD45RA, mouse Ly-6G, mouse TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leucocyte antigen (HLA) DR (HLA-DR) and CD235a (glycophorin A).These antigenic hemopoietic stem cells of not expressing level of signification are commonly referred to as lineage negative (Lin ^-).General other surface antigen of expressing of human hematopoietic stem cell such as CD31, CD34, CD117 (c-kit) and/or CD133.General other surface antigen of expressing of mouse hemopoietic stem cell such as CD34, CD117 (c-kit), Thy-1 and/or Sca-1.

The present invention is provided at " pedigree surface antigen " the isolating hemopoietic stem cell (Lin) of not expressing level of signification on its cell surface.This cell is called " lineage negative " or " Lin in this article ^-" hemopoietic stem cell.Specifically, the present invention provides the Lin that contains endothelial progenitor cells (EPC) ^-Hemopoietic stem cell crowd (Lin ^-HSC), it can penetrate in the developmental blood vessel, is differentiated to form vascular endothelial cell then.Preferably, isolating Lin ^-HSC crowd is present in substratum such as the PBS (PBS).

The statement of using in this paper and the appended claim " adult " is used in reference to marrow, comprises the isolating marrow in birth back, promptly with embryo's isolating marrow from childhood and adult individuality on the contrary.Term " adult Mammals " refers to childhood and the Mammals that grows up fully this two.

Hemopoietic stem cell (the Lin of isolating, mammiferous, lineage negative of the present invention ^-HSC) crowd comprises endothelial progenitor cells (EPC).Preferably, isolating Lin ^-HSC crowd comprises wherein the mammalian cell at least about 20% cell expressing surface antigen CD31, and surface antigen CD31 is present on the endotheliocyte usually.In another embodiment, at least about 50%, more preferably at least about 65%, most preferably at least about 75% cell expressing CD31.Preferably, at least about 50% Lin of the present invention ^-HSC crowd's cell preferred expression beta 2 integrin alpha 6 antigens.

At a preferred mouse Lin ^-In HSC crowd's embodiment, the cell expressing CD31 antigen at least about 50%, cell expressing CD117 (c-kit) antigen at least about 50%.Preferably, at least about 75%, more preferably from about 81% Lin-HSC cell expressing surface antigen CD31.In another preferred mouse embodiment, the cell expressing surface antigen CD117 at least about 65%, more preferably from about 70%.A particularly preferred embodiment of the present invention is following mouse Lin ^-HSC crowd, wherein about 50% to about 85% cell expressing surface antigen CD31, about 70% to about 75% cell expressing surface antigen CD117.

Another preferred embodiment is that wherein cell is the negative people Lin of CD133 ^-HSC crowd, wherein at least about 50% cell expressing CD31 surface antigen, cell expressing beta 2 integrin alpha 6 antigens at least about 50%.Another preferred embodiment is that wherein cell is CD133 male people Lin again ^-HSC crowd wherein is less than about 30% cell expressing CD31 surface antigen, is less than about 30% cell expressing beta 2 integrin alpha 6 antigens.

Isolating Lin of the present invention ^-When HSC crowd injected the mammalian species eye in vitreum, selectivity target star spongiocyte also mixed in the new vessel system, and said mammalian species for example is mouse or people, and said cell is by separating in the said mammalian species.

Isolating Lin of the present invention ^-HSC crowd is contained endothelial progenitor cells, and it is divided into endotheliocyte and in retina, produces blood vessel structure.Specifically, Lin of the present invention ^-HSC crowd is used to treat retinal neovascularization and retinal vessel degenerative disease, and is used for the repairing retina blood vessel injury.Lin of the present invention ^-The HSC cell also promotes the neurone rescue in the retina, and impels anti-apoptotic genes expression to raise.Be surprisingly found out that adult Lin of the present invention ^-HSC cell even can in suffering from the Reconstruction in Sever Combined Immunodeciency of retinal degeneration (SCID) mouse, suppress retinal degeneration.The normal mouse retina is mainly retinal rod, but uses Lin ^-After the HSC treatment almost all is the cone by the rescue cell.In addition, Lin ^-HSC crowd can be used for treating the retina defective in the nascent Mammals eye, and said nascent Mammals is for example for suffering from the Mammals of oxygen induction type retinopathy or retinopathy of prematurity.

The present invention also provides a kind of method of treating the Mammals eye illness; It comprises the lineage negative hematopoietic stem cells crowd contain endothelial progenitor cells by separating in the Mammals marrow, and isolating stem cell is gone in the Mammals eye with the amount intravitreal injection that is enough to suppress disease.That the inventive method is used in is nascent, treat eye illness in Mammals childhood or that grow up fully, for example retinal degenerative disease, retinal vessel degenerative disease, ischemic retinopathy, angiorrbagia, vascular leakage and choroidopathy.The instance of these diseases comprises AMD (ARMD), diabetic retinopathy (DR), intends ocular histoplasmosis (POHS), retinopathy of prematurity (ROP), sicklemia and retinitis pigmentosa and retina injury.

Being expelled to stem cell number in the eye is enough to suppress the morbid state of eyes.For example, cell count can effectively repair the eyes retina damage, stablize retinal neovascularization system, ripe retinal neovascularization system and prevent or repair vascular leakage and angiorrbagia.

Useful gene on the available treatment, the gene of the gene of the angiogenesis inhibitor albumen of for example encoding (the gene therapy that is used for eye) and coding neurotrophy material (being used to strengthen neurone rescue effect), transfection Lin of the present invention based on cell ^-HSC crowd's cell.

Cells transfected can comprise any gene useful to retina obstacle treatment in treatment.In a preferred embodiment, the Lin of transfection of the present invention ^-HSC comprises the gene of efficient coding angiogenesis inhibitor peptide; This angiogenesis inhibitor peptide comprises albumen or its fragment; The fragment of TrpRS or its angiogenesis inhibitor for example; T1 and the T2 fragment of the TrpRS that for example in u.s. patent application serial number 10/080,839 common all, common pending trial, is detailed, the disclosure of this patented claim is attached among this paper by reference.The transfection Lin of coding angiogenesis inhibitor peptide of the present invention ^-HSC is used to treat and the relevant retinopathy of abnormal vascular growth, for example diabetic retinopathy and similar disease.Lin ^-HSC preferably is people's cell.

In a further preferred embodiment, transfection Lin of the present invention ^-HSC contains the gene of effective coding neurotrophy material, and said neurotrophy material for example is NGFF, neurotrophin-3, neurotrophin-4, neurotrophin-5, CNTF, retinal pigment epithelium deutero-neurotrophic factor, rhIGF-1, glial cell-line deutero-neurotrophic factor, brain derived neurotrophic factor etc.This type neurotrophic Lin ^-HSC is used in and promotes neurone rescue, the damage of treatment retina neural etc. in the retina nerve degeneration disease (for example glaucoma and retinitis pigmentosa).Reported already that the CNTF implant was used to treat retinitis pigmentosa (referring to Kirby etc., 2001, Mol Ther.3 (2): 241-8; Farrar etc., 2002, EMBO Journal 21:857-864).It is reported, brain derived neurotrophic factor in impaired ganglia retinae, regulate growth related gene (referring to Fournier etc., 1997, J.Neurosci.Res.47:561-572).It is reported that glial cell-line deutero-neurotrophic factor delays the sight sensor sex change (referring to McGee etc., 2001, Mol Ther.4 (6): 622-9) in retinitis pigmentosa.

The present invention also provides a kind of method of from Mammals marrow, separating the lineage negative hematopoietic stem cells that contains endothelial progenitor cells.This method needs following steps: (a) extract marrow from the adult Mammals; (b) from marrow, separate multiple monocyte; (c) use the pedigree colony antibody to one or more antigenic biotin-conjugated in pedigree surface to come the mark monocyte, preferred pedigree surface antigen is selected from: CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (mouse), TER-119 (mouse), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leucocyte antigen (HLA) DR (HLA-DR) and CD235a (glycophorin A); (d) from multiple monocyte, remove the positive monocyte of said one or more pedigree surface antigens, and reclaim the lineage negative hematopoietic stem cells crowd contain endothelial progenitor cells, preferred wherein at least about 20% cell expressing CD31.

When from adult's marrow, separating Lin ^-During HSC; Preferably, use the pedigree colony antibody labeling monocyte of the biotin-conjugated that is directed against pedigree surface antigens c D2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 (B7.2) and CD235a.When from the adult mouse bone marrow cells, separating Lin ^-During HSC, preferably, with the pedigree colony antibody labeling monocyte of the biotin-conjugated that is directed against pedigree surface antigens c D3, CD11, CD45, Ly-6G and TER-119.

In a kind of preferred method, said cellular segregation is from the marrow of being grown up, and further separates through the CD133 pedigree.Separation of human Lin ^-A kind of preferred method of HSC comprises following additional step: with the CD133 antibody labeling monocyte of biotin-conjugated, and reclaim CD133 male Lin ^-HSC crowd.Usually, this type of cell expressing CD31 less than about 30%, this type of cell expressing beta 2 integrin alpha 6 less than about 30%.CD133 male people Lin of the present invention ^-HSC crowd is being injected into the angiogenesis position that but the targeting peripheral local asphyxia induces when not experiencing in the eye that blood vessel takes place.

Separation of human Lin ^-The another kind of preferred method of HSC comprises following additional step: with the CD133 antibody labeling monocyte of biotin-conjugated, remove the CD133 positive cell and reclaim the negative Lin of CD133 ^-HSC crowd.Usually, this type of cell expressing CD31 at least about 50%, this type of cell expressing beta 2 integrin alpha 6 at least about 50%.The people Lin that CD133 of the present invention is negative ^-HSC crowd can infiltrate in the developmental vascular system when not experiencing in the eye that blood vessel takes place being injected into.

The present invention also provides the method for treatment ocular angiogenesis generation disease, and this method is through giving the Lin of transfection of the present invention during said cell intravitreal injection is pleasing to the eye ^-The HSC cell.The Lin of this transfection ^-The HSC cell comprises the Lin that goes up useful gene (gene of for example encode angiogenesis inhibitor or neurotrophic gene product) transfection with treatment ^-HSC.Preferably, the Lin of transfection ^-The HSC cell is people's cell.

Preferably, at least about 1 * 10 ⁵Individual Lin ^-The Lin of HSC cell or transfection ^-The HSC cell suffers from the Mammals eye of retinal degenerative disease through intravitreal injection.The other factors that the those of ordinary skill of wanting the injected cells number to can be depending on seriousness, Mammals age and the retinal diseases treatment field of retinal degeneration is known easily.Lin ^-HSC can give with single dose or multiple doses in for some time, and this is by clinician's decision of being responsible for treatment.

Lin of the present invention ^-HSC is used to treat retina injury and the retina defective that relates to retinal vessel system outage or sex change or retina nerve degeneration.People Lin ^-HSC also can be used for producing heredity and goes up identical clone, and promptly clone is used for reproducibility or treatment of restoration nature retinal blood guard system, and is used for treatment or improves the retinal neurons sex change.

Embodiment

Embodiment 1. cellular segregation and enrichment; Mouse Lin ^-The preparation of HSC crowd A and B

General method.All interior evaluatings are all carried out according to nurse of NIH laboratory animal and instruction manual, and all assessment processes all obtain Scripps institute (TSRI, La Jolla, CA) approval of the tending of animals and the council of use.From B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB/NJ (rd/rd mouse) or Balb/cBYJ adult mouse (The JacksonLaboratory, ME) the middle medullary cell that extracts.

Then, through using HISTOPAQUE

(density gradient separation MO) is come separating monocytic cell to the ficoll gradient for Sigma, St.Louis, and is used in and is used for Lin in the mouse ^-Pedigree colony antibody (CD45, CD3, Ly-6G, CD11, TER-119, Pharmingen, San Diego, CA) mark of the biotin-conjugated of selecting.Use magnetic separating apparatus (AUTOMACS ^TMSorter, Miltenyi Biotech, Auburn is CA) from Lin ^-Separate the positive (Lin of pedigree among the HSC ⁺) cell and removing.The Lin that contains endothelial progenitor cells that is obtained ^-HSC crowd uses following antibody to use FACS ^TMThe Calibur flow cytometer (Becton Dickinson, Franklin Lakes NJ) further characterize: and the Sca-1 that PE-puts together, c-kit, KDR and CD31 (Pharmingen, SanDiego, CA).The Tie-2-GFP medullary cell is used to characterize Tie-2.

For results adult mouse endotheliocyte, from the ACTbEGFP mouse, take out mesentery tissue through surgical operation, and place collagenase (Worthington, Lakewood, NJ) in the digestion tissue, use 45 μ m filters to filter then.Collect circulation filtrating and with endothelial growth substratum (Clonetics, San Diego, CA) incubation.Through observe modal cobblestone-appearance outward appearance, with CD31mAb (Pharmingen) dyeing and inspection culture at MATRIGEL ^TM(NJ) the middle pipe spline structure that forms is confirmed the endothelium proterties to matrix for Beckton Dickinson, Franklin Lakes.

Mouse Lin ^-HSC crowd A.From the ACTbEGFP mouse, extract medullary cell through above-mentioned general method.Characterize Lin through the FACS flow cytometer ^-The CD31 of HSC cell, c-kit, Sca-1, Flk-1 and Tie-2 cell-surface antigens mark.The result is shown in Fig. 1 (c).About 81% Lin ^-HSC demonstrates the CD31 mark, about 70.5% Lin ^-HSC demonstrates the c-kit mark, about 4% Lin ^-HSC demonstrates the Sca-1 mark, about 2.2% Lin ^-HSC demonstrates the Flk-1 mark, about 0.91% Lin ^-HSC demonstrates the Tie-2 mark.On the contrary, isolating Lin from these medullary cells ⁺HSC has remarkable different cells mark spectrum (that is CD31:37.4%; C-kit:20%; Sca-1:2.8%; Flk-:0.05%).

Mouse Lin ^-HSC crowd B.From Balb/C, ACTbEGFP and C3H mouse, extract medullary cell through above-mentioned general method.Analyze Lin ^-Cell surface marker in the HSC cell (Sca-1, KDR, c-kit, CD34, CD31 and multiple integrin: α 1, α 2, α 3, α 4, α 5, α 6, α _M, α _V, α _X, α _IIb, β ₁, β ₄, β ₃, β ₄, β ₅And β ₇) have a situation.The result is as shown in table 1.

Table 1.Lin ^-The characteristic of HSC crowd B

Embodiment 2. gives cell in the vitreum in mouse model

In the mouse eyelid, do an eyelid crack with sharp blade, expose P2 to P6 eyeball.Use one No. 33 (Hamilton, Reno, the NV) syringe of syringe needle, intravitreal injection lineage negative HSC crowd of the present invention A (has an appointment 10 at about 0.5 μ l to about 1 μ l cell culture medium ⁵Individual cell).

Embodiment 3.EPC transfection

Laboratory manual according to manufacturer utilizes FuGENE ^TM(Roche, Indianapolis is IN) with the T2 fragment of coding TrpRS, enclose His again for 6 transfection reagents ₆The DNA of mark (SEQ ID NO:1, Fig. 7) transfection mouse Lin ^-HSC (crowd A).With Lin ^-(every ml about 10 for the HSC cell ⁶Cell) is suspended in and contains STEMCELLFACTOR (PeproTech, Rocky Hill, opti-MEM NJ) Substratum (Invitrogen, Carlsbad, CA) in.Add DNA (about 1 μ g) and FuGENE reagent (about 3 μ l) then, with mixture about 18 hours of about 37 ℃ of incubations.Behind the incubation, clean and collecting cell.The transfection efficiency that confirms this system through facs analysis is about 17%.Confirm that through western blotting T2 produces.His ₆The aminoacid sequence of the T2-TrpRS of mark is shown in the SEQ ID NO:2 of Fig. 8.

Embodiment 4. immunohistochemistries and copolymerization are burnt to be analyzed

Collect the mouse retina in different time points, for self-contained or frozen section film-making are prepared.For self-contained, use fixedly retina of 4% Paraformaldehyde 96, in 50% foetal calf serum (FBS) and 20% normal goats serum, sealed one hour in room temperature.Retina resists detection through an anti-processing and with two.What use one anti-ly is: and the former protein I V of anticol (Chemicon, Temecula, CA), anti-β-gal (Promega; Madison, WI), anti-GFAP (Dako Cytomation, Carpenteria; CA), anti--α-smooth muscle actin (α-SMA, Dako Cytomation).Use two anti-put together Alexa 488 or 594 fluorescent marks (Molecular Probes, Eugene, OR).(Bio-Rad, Hercules CA) obtain image to utilize MRC 1024 Laser Scanning Confocal Microscopes.Utilize LASERSHARP

software (Bio-Rad) to create 3-D view, to check the growth of three different blood vessel layers in the self-contained retina.The GFP pixel intensity difference that the GFP that utilization is picked out by Laser Scanning Confocal Microscope (eGFP) strengthens between mouse and GFAP/wtGFP mouse is created 3D rendering.

Retinal vessel generation quantitatively determined in the body in embodiment 5. mouse

Analyze for T2-TrpRS, from elementary clump of mouse retina three-dimensional image reconstruction and dark clump.Elementary clump is divided into two types: the vascular development of normal development or interruption.The classification that the deep vascular development suppresses is analyzed based on vaso-inhibitory per-cent; Comprise following standard: the inhibition fully that dark clump forms is labeled as " fully "; Normal blood vessels growth (contain and be less than 25% inhibition) is labeled as " normally ", and remaining is labeled as " part ".For the rescue data of rd/rd mouse, use the 10x lens to obtain four individual region of dark clump in each self-contained retina.Calculate every width of cloth image the vascular system length overall, sum up and organize between relatively.In order to obtain accurate information, with Lin ^-HSC is injected in the eye of mouse, and with Lin ⁺HSC is expelled in the another eye of same mouse.The contrast retina of injection is not taken from a brood of animal.

Embodiment 6. adult retina injury mouse models

The use diode laser (150mW, 1 second, 50mm) or with No. 27 syringe needle machineries puncture the mouse retina, set up laser and scar model.In wound back 5 days, utilize method injection cell in the vitreum.Gather the mouse eyes after 5 days.

The neurotrophic rescue of embodiment 7. retinal degenerationes

Adult mouse bone marrow lineage negative hematopoietic stem cells (Lin ^-HSC) in the retinal degeneration mouse model, have vascular nutrition property and neurotrophic rescue effect.The right eye intravitreal injection of mouse contained about 10 for about 0.5 milliliter 10 day age ⁵Individual Lin of the present invention ^-HSC, the number that has situation and neurone layer nuclear of 2 months postevaluation retinal blood guard systems.The Lin of the about similar number of left eye injection of same mouse ⁺HSC is as contrast, and carries out similar evaluation.As shown in Figure 9, at Lin ^-In the eye of HSC treatment, retinal vessel shows near normal, and inner nuclear layer is approximate normal, outer nuclear layer (ONL) 3 to 4 stratum nucleares of having an appointment.On the contrary, offside Lin ⁺The eye that HSC handles has the middle retina vascular lamina of an obvious atrophy, the outer retina vascular lamina of a complete atrophy; The obvious atrophy of inner nuclear layer, the outer nuclear layer completely dissolve.This shows particularly evidently in mouse 3 and mouse 5.In mouse 1, do not see the rescue effect, this situation be present in about 15% by the injection mouse in.

When using electroretinogram (ERG) evaluation sight function, when observing blood vessel and neural rescue (mouse 3 and 5), observe positive ERG and recover.(mouse 1) do not observe positive ERG when having the rescue of blood vessel and neurone.Regression analysis curve shows shown in Figure 10 Lin of the present invention ^-Dependency between rd/rd mouse eye medium vessels that HSC produces and the neurotrophy rescue.For middle vascular system type (r=0.45) and dark vascular system (r=0.67), observe the dependency between the reparation of neurone (y-axle) and blood vessel (x-axle).

Figure 11 is presented at Lin ⁺Has no the significant correlation property on the statistics between blood vessel that HSC produces and the neurone rescue.To the blood vessel rescue quantitatively, data are listed in Figure 12.Mouse data when back 1 month (1M) of injection that Figure 12 shows, 2 months (2M) and 6 months (6M) show, compare with the length of vessel (Bai Zhu) in the eye that is untreated of same mouse, and use Lin of the present invention ^-Length of vessel in the eye that HSC handles (black post) has remarkable increase, particularly back 1 month of injection during with 2 months.At injection Lin ^-HSC or Lin ⁺Behind the HSC about two months, through the amount of the appraising and deciding neurotrophic rescue effect in counting inner nuclear layer and the outer nuclear layer.The result lists in Figure 13 and 14.

Embodiment 8. people Lin ^-HSC crowd

From adult healthy volunteers, extract medullary cell through above-mentioned general method.Then through using HISTOPAQUE

ficoll gradient (Sigma; St.Louis, density gradient separation MO) is come separating monocytic cell.In order from people's myelomonocyte, to separate Lin ^-HSC crowd is used for magnetic separation system (AUTOMACS with the pedigree colony antibody of following biotin-conjugated ^TMSorter, Miltenyi Biotech, Auburn, CA): CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a (Pharmingen).

Express people Lin based on CD133 ^-HSC crowd is further divided into two subgroups.Cell is with the CD133 antibody labeling of biotin-conjugated, and be divided into CD133 positive with CD133 feminine gender subgroup.

Embodiment 9. administration of human and mouse cell in the vitreum in the retinal degeneration mouse model

C3H/HeJ, C3SnSmn.CB17-Prkdc SCID and rd10 mouse species are as the retinal degeneration model.(TheJackson Laboratory is the homozygote of retinal degeneration 1 (rd1) sudden change Maine), and said sudden change is the sudden change that causes the serious retinal degeneration of early onset thereof for C3H/HeJ and C3SnSmn.CB17-Prkdc SCID mouse.This sudden change is arranged in the exon 7 of the Pde6b gene of coding retinal rod sight sensor cGMP phosphodiesterase β subunit.This sudden change in this gene is found in the people patient that autosomal recessive retinitis pigmentosa (RP) arranged.C3SnSmn.CB17-Prkdc SCID mouse still is Reconstruction in Sever Combined Immunodeciency spontaneous mutation (Prkdc SCID) homozygote, is used for the experiment of people's cell transfer.Retinal degeneration in the rd10 mouse is caused by the sudden change in the exons 13 of Pde6b gene.This also is that a kind of outbreak is back than rd1/rd1, the retinal degeneration clinical correlation RP model slighter than rd1/rd1.All are estimated all and implement according to nurse of NIH laboratory animal and instruction manual, and all methods all obtain Scripps institute (TSRI, La Jolla, CA) approval of the tending of animals and the council of use.

In the mouse eyelid, do an eyelid crack with sharp blade, expose P2 to P6 eyeball.Use one No. 33 (Hamilton, Reno, the NV) syringe of syringe needle, (about 0.5 μ l is to about 10 of about 1 μ l cell culture medium with the lineage negative HSC cell of mouse crowd A or crowd C ⁵Individual cell) intravitreal injection is gone in the little rathole.For people's cell of making injection visible, before injection with dyestuff (Cell tracker green CMFDA, Molecular Probes) labeled cell.

Collect retina in different time points, and fix, then in 50%FBS/20%NGS, sealed one hour in room temperature with 4% Paraformaldehyde 96 (PFA) and methyl alcohol.For dyeing retinal blood guard system,, follow two anti-(Molecular Probes, Eugene, Oregon) incubations of puting together with Alexa 488 or 594 with retina and anti-CD31 antibody (Pharmingen) and the former protein I V of anticol antibody (Chemicon) incubation.With four radioactivity relaxing incisions tiling retinas, to obtain self-contained sheet articles.Retinal vessel intermediate plexus or dark clump (referring to Dorrell etc.; 2002 InvestOphthalmol.Vis.Sci.43:3500-3510) vascular system imagery exploitation RadianceMP2100 confocal microscope in and LASERSHARP software (Biorad; Hercules California) obtains.Be quantitative vascular system; Select four isolated areas (900 μ m * 900 μ m) at random from the middle portion of middle vascular lamina or dark vascular lamina, use LASERPIX

analysis software (Biorad) to measure the blood vessel length overall.The length overall in these four zones in the same clump is used for further analysis.

For making frozen section, embedding retina plain film again.Retina places 4% PFA to spend the night, then with 20% sucrose incubation.Retina is embedded in optimal cutting temperature compound (OCT:Tissue-Tek; Sakura FineTech, Torrance, CA) in.Frozen section (10 μ m) is containing nuclear dyestuff DAPI (Sigma-Aldrich, St.Louis, hydration again among PBS Missouri).Obtain the DAPI mark nuclear image in three the different visuals field (280 μ m are wide, unbiased sampling) that contain in optic nerve head and the amphiblestroid single section of complete perimeter through confocal microscope.The check figure order of the ONL that is arranged in three independent visuals field of a section is counted and sued for peace, to analyze.Carry out simple linear regression analysis, to detect vascular system length and the relation between the cell check figure among the ONL in the dark clump.

After dark adatpation whole night, through peritoneal injection 15 μ g/gm ketamines and 7 μ g/gm xylazine anesthetized mices.Use gold ring Corneal electrode together with mouth reference electrode and tail ground-electrode, (1% Tropintran) is from the anterior corneal surface record electroretinogram ERG (ERG) of every eye behind platycoria.(Quincy MA) produces stimulation for PS33 Plus, Grass Instruments with the Grass photic stimulator that is fixed on the highly reflective Ganzfeld dome outside.Until photic stimulator maximum permissible value (0.668cd-s/m ²) brightness range in, to short wavelength (Wratten 47A; λ _Max=470nm) flash of light record retinal rod reaction.To reaction signal amplify (CP511AC magnifying glass, Grass Instruments), digitizing (PCI-1200, National Instruments, Austin, TX) and Computer Analysis.Every mouse is with by the ERG of the eye of treatment and the untreated ophthalmic record interior mark as himself.With nearly 100 times scanning is average, as weak signal.Reaction numeral by the treatment eye deducts the not average response of treatment eye, and this signal difference is used to indicate functional rescue.

Use microarray analysis to estimate Lin ^-The retina genetic expression of HSC target.Use Lin ^-HSC or CD31-HSC injection P6rd/rd mouse.Inject back 40 days detachment of retina (this time after injection is pointed out the rescue that shows visible retinal vessel and sight sensor layer) in the substratum that does not have the RNA enzyme with these mouse.Analyze through self-contained that each is amphiblestroid 1/4, to guarantee normal HSC target and to have obtained vascular system and neuroprotective.Use TRIzol (Life Technologies, Rockville, MD), the retina purifying RNA that injects by success of the imitative RNA separation scheme of phenol/atmosphere.RNA is hybridized the chip to Affymetrix Mu74Av2; Use GENESPRING

software (SiliconGenetics; Redwood City, CA) analyzing gene is expressed.The people of purifying or mouse HSC intravitreal injection are gone in the P6 mouse.When P45, peel off retina and be merged into: 1) injected the mouse retina that the quilt of people HSC is saved with the lower section; 2) injected the mouse retina of not saved of people HSC; And 3) injected the mouse retina that the quilt of mouse HSC is saved; Be used for purifying RNA, and hybridize to human specific U133A Affymetrix chip.Using GENESPRING

software to differentiate is expressing more than the background value and in the retina of people HSC rescue, is having the gene than high expression level.Then in these genes of separate analysis the probe of each to express spectra, and with the normal people U133A microchip experimental model contrast of using dChip, confirming the specific hybrid of human species, and eliminate because the false positive that species hybridization produces.

Figure 21 table has shown the flow cytometer data, and these data have compared CD31 and beta 2 integrin alpha 6 surface antigens at the positive (DC133 of CD133 of the present invention ⁺) and the negative (CD133 of CD133 ^-) people Lin ^-Expression on the HSC crowd.Left figure has shown the flow cytometry dot chart.The figure of central authorities is a histogram with right figure, has shown the expression level of specific antibody on the cell mass.The Y axle is represented event number, and the X axle shows strength of signal.Hollow histogram is the isotype IgG control antibodies that shows non-specific background dye level.Solid histogram has shown the expression level of specific antibody on the said cell mass.Move to the fluorescent signal and the above antibody expression of background level of the solid histogram representative increase on hollow (contrast) histogram right side.The protein expression difference on the cell has been represented in the contrast of solid histogram peak position between two cell masses.For example, CD31 is at CD133+ of the present invention and CD133 ^-All more than background, express on the cell; But, and CD133 ^-The faciation ratio, the cell of the CD31 of expression lower level is more in the CD133+ cell mass.Data are obvious thus, and CD31 is expressed between two crowds and changes, and α 6 integrin expression major parts are limited to Lin ^-Therefore cell among the crowd can be used as the affinity tag of the cell with vascular nutrition property and neurotrophic rescue function.

As the positive Lin negative of CD133 with CD133 ^-When HSC subgroup intravitreal injection is gone into to come into being in the eye of SCID mouse,, observe growing the at utmost infiltration (referring to Figure 21, figure below) of medium vessels system for not only expressing CD31 but also expressing the negative subgroup of CD133 of beta 2 integrin alpha 6 surface antigens.The positive subgroup of CD133 (Figure 21, last figure) of not expressing CD31 or beta 2 integrin alpha 6 seems the angiogenesis position that the targeting peripheral local asphyxia induces, but be injected into just experience angiogenesis in the time do not have this phenomenon.

Using has saved with the immunohistochemical methods methods analyst and the retina that do not saved of specific antibody to retinal rod opsin or cone opsin.Same eye to being used for the ERG record that Figure 17 lists is carried out retinal rod opsin or the analysis of cone opsin.In the wild-type mice retina; The sight sensor less than 5% that exists is the cone (Soucy etc.; 1998; Neuron21:481-493), the immunohistochemical staining collection of illustrative plates that is observed with red/green cone opsin or with the retinal rod Visual purple meets this percentage of cone cell shown in Figure 25 (A) or Figure 25 (B).Robert doctor Molday by British Columbia university provides to the specific antibody of retinal rod Visual purple (rho4D2), use as discussed previously (Hicks etc., 1986, Exp.Eye Res.42:55-71).To the cone red/the specific rabbit antibody of green opsin is available from Chemicon (AB5405), use according to manufacturer's explanation.

Embodiment 10. gives the mouse cell in the vitreum in the mouse model of oxygen induction type retinal degeneration

P7 to P12 makes newborn wild-type C57B16 mouse be exposed to oxygen environment (75% oxygen) between day after the birth in oxygen induction type retinal degeneration (OIR) model.Figure 22 has shown by the normal nascent back vascular development in the C57B16 mouse during the P0 to P30.When P0, can only around discus nervi optici, observe the top layer blood vessel that has just germinated.In ensuing several days, elementary top layer network extends to periphery, arrives the neighboring during day at P10.Between P7 and P12, secondary (deeply) burst is educated.During to P17, top layer and deep-level blood vessel network (Figure 22, illustration) widely appear.In later time, reinvent together with the growth of the 3rd (centre) of blood vessel layer, until greatly about P21 the time, form mature structure.

On the contrary, in the OIR model, be that P7-P12 is exposed to after 75% oxygen, the regular turn of incident is by serious upset (Figure 23).Adult mouse Lin of the present invention ^-HSC crowd's intravitreal injection when P3 is gone in the eye of mouse, and this mouse carries out OIR subsequently, and another eye injection PBS or CD31 negative cells are as contrast.Figure 24 shows, Lin of the present invention ^-HSC crowd can reverse the Denaturation of hyperoxia level in developmental mouse retina.When P17, in the eye of handling, observe the top layer and the deep layer retinal blood guard system of growing fully, and in the contrast eye, demonstrate a large amount of avascular areas territory, being actually does not have deep-level blood vessel (Figure 24).About 100 the mouse eyes in the OIR model have been observed.Lin of the present invention 58% ^-HSC handles and observes normal blood vessels formation in the eye, by contrast, uses CD31 ^-The contrast eye that cell is handled is 12%, and the contrast eye that uses PBS to handle is 3%.

Result and discussion

The mouse retinal vessel is grown; Eye blood vessel generation model.The mouse eyes provide a kind of generally acknowledged being used to study the model that the Mammals retinal vessel is grown (for example human retina vascular development).During the phylogeny of mouse retinal vessel, the retinal vessel that local asphyxia induces is to grow with the tight associating mode of star spongiocyte.These neuroglia elements move to along ganglion-cell layer from discus nervi optici on the human foetus or newborn rodentine retina of third trimester pregnancy, and expand radially.Along with the mouse retinal vessel is grown, endotheliocyte utilizes this star spongiocyte template of having set up to confirm retinal vessel pattern (referring to Fig. 1 (a and b)).Fig. 1 (a and b) illustrates developmental mouse retina synoptic diagram.Figure (a) illustrates the growth that is superimposed upon the elementary clump (scheming upper left concealed wire) on the star spongiocyte template (bright line), and (b) has described the subordinate phase that retinal vessel forms.In Fig. 1, GCL represents ganglion-cell layer; Clump layer in the IPL representative; INL represents inner nuclear layer; The outer clump layer of OPL representative; ONL represents outer nuclear layer; RPE represents retinal pigment epithelium; ON represents optic nerve; And P represents periphery.

At birth, the retinal blood guard system is actually non-existent.To be born back 14 days (P14), retina has developed into complicated elementary (table) and secondary (deeply) layer retinal vessel, forms with vision to conform to.During beginning, the radial nipple surrounding blood vessel of looking is radial growth to periphery on already present star spongiocyte network, forms through the capillary vessel clump and becomes interconnection progressively.These blood vessels are grown as monolayer in nerve fiber during to P10 (Fig. 1 (a)).Between P7-P8, the from then on elementary clump of side accessory branch begins growth, penetrates retina to outside plexiform layers, and they form secondary or deep layer retina clump there.During to P21, whole reticulated structure has experienced extensive reconstruction, forms third stage clump or intermediate plexus (Fig. 1 (b)) at the inner nuclear layer internal surface.

Because some reasons, newborn mice retinal vessel generation model can be used for studying the HSC effect of a blood vessel between the emergence period.In this physiology correlation model,, the endogenous blood vessel has a large amount of star spongiocyte templates before occurring, and this makes can estimate cell-cell-targeting effect in angiogenic process.In addition, known this links up and reproducible newborn retinal vessel process is that hypoxemia drives, and has similarity with the acting retinal diseases of many wherein known local asphyxias thus.

By marrow enrichment endothelial progenitor cells (EPC).Though extensively estimated the cell surface marker expression on the EPC crowd in being present in the HSC prepared product, the mark of single-minded identification EPC still is difficult to confirm.For enrichment EPC, with hematopoietic lineage mark positive cell (Lin ⁺), promptly bone-marrow-derived lymphocyte (CD45), T lymphocyte (CD3), granulocyte (Ly-6G), monocyte (CD11) and red corpuscle (TER-119) are removed from the mouse bone marrow cells monocyte.With the further enrichment EPC of Sca-1 antigen.The Lin of contrast intravitreal injection equivalent ^-Sca-1 ⁺Cell or Lin ^-The result who obtains behind the cell does not detect difference between two groups.In fact, when only injecting Lin ^-Sca-1 ^-During cell, observe much bigger infiltration far away to the growth medium vessels.

Enrichment has the Lin of the present invention of EPC based on functional examination ^-HSC crowd.In addition, Lin ⁺HSC crowd and Lin ^-HSC crowd shows quite differently on function.Also estimated the epi-position (based on the external The Characteristics of previous report) of the EPC that is usually used in differentiating each several part.Although none and Lin in these marks ^-The single-minded association of part, but same Lin ⁺HSC partly compares, Lin ^-Institute among the HSC is underlined all to increase about 70% to about 1800% (Fig. 1 (c)).The figure c of Fig. 1 has explained bone marrow Lin ⁺HSC and Lin ^-The flow cytometry characteristic of HSC isolated cell.The top row of Fig. 1 (c) has shown that the hemopoietic stem cell dot chart of non-antibody labeled cell distributes.R1 has confirmed the painted quantitative gate of positive PE-district; GFP-is positive in the R2 indication.Middle line has shown Lin ^-The dot chart of HSC, end row has shown Lin ⁺The dot chart of HSC.The C57B/6 cell is puted together antibody labeling with the PE-to Sca-1, c-kit, Flk-1/KDR, CD31.The Tie-2 data derive from the Tie-2-GFP mouse.Per-cent in the dot chart angle is represented total Lin ^-HSC or Lin ⁺The percentage ratio of positive labeled cell among the HSC crowd.What is interesting is that EPC mark of generally acknowledging such as the expression of Flk-1/KDR, Tie-2 and Sca-1 are all very poor, therefore be not used in further fractional separation.

The HSC Lin of intravitreal injection ^-Cell contains the target star spongiocyte and infiltrates the EPC of developmental retinal vessel.In order to measure the Lin of intravitreal injection ^-Whether HSC can the amphiblestroid particular cell types of target, utilize the star spongiocyte template and participate in retinal vessel and take place, will be from Lin of the present invention ^-About 10 of HSC compsn ⁵Individual cell or separation are from the Lin of adult (GFP or LacZ transgenic) mouse bone marrow cells ⁺HSC cell (contrast, about 10 ⁵Individual cell) is injected in the little rathole of birth back 2 days (P2).Inject back four days (P6), derive from the Lin of the present invention of GFP or LacZ transgenic mice ^-Many cell attachment of HSC compsn and have the characteristic elongation outward appearance (Fig. 2 (a)) of endotheliocyte on retina.Fig. 2 has explained Lin ^-Cell moves in the developmental mouse retina.Shown in Fig. 2 (a), inject the eGFP+Lin of back four days (P6) intravitreal injections ^-HSC adheres on retina and breaks up.

In amphiblestroid a lot of zones, the GFP express cell is arranged with the pattern that meets following star spongiocyte, and similar with blood vessel.These fluorocytes were observed (Fig. 2 (b)) before endogenic, developmental blood vessel network.On the contrary, has only minority Lin ⁺HSC (Fig. 2 (c)) or adult mouse mesentery endotheliocyte (Fig. 2 (d)) are attached to retinal surface.In order to confirm Lin from injection ^-Whether HSC crowd's cell also can be attached on the retina of setting up blood vessel, and we are with Lin ^-The HSC compsn is injected in the adult eye.What is interesting is, do not observe cell attachment on retina or infiltrate (Fig. 2 (e)) in the existing normal retinal vessel.This shows Lin of the present invention ^-The HSC compsn does not destroy the blood vessel that normal development is accomplished, can exception throw vascularization in the retina of normal development.

In order to confirm the Lin of injection of the present invention ^-Relation between HSC compsn and the retina star spongiocyte is used transgenic mice, and it expresses the green fluorescent protein (GFP) of glial fibrillary acidic protein (GFAP, the affinity tag of star spongiocyte) and promoters driven.To having injected Lin from the eGFP transgenic mice ^-The retina of these GFAP-GFP transgenic mices of HSC checks, shows that eGFP EPC and the existing star spongiocyte injected locate (Fig. 2 (f-h), arrow place) altogether.Observe eGFP+Lin ^-The process of HSC consistent with following star spongiocyte reticulated structure (arrow place, Fig. 2 (g)).Inspection to these shows that the labeled cell of being injected only is attached on the star spongiocyte; Still do not have in the P6 mouse retina of endogenous blood vessel in the retina periphery, observe institute's injected cells attached to these as yet not on the star spongiocyte in the vascularization zone.Surprisingly, on the definite position that normal retinal vessel will be grown subsequently in the retina deep layer, observe the labeled cell of being injected (Fig. 2 (i), arrow place).

In order to confirm the Lin of injection of the present invention ^-Whether HSC stably penetrates in the developmental retinal blood guard system, at several time point inspection retinal vessels after a while.When P9 (injecting back 7 days), Lin ^-HSC has penetrated into CD31 ⁺In the structure (Fig. 2 (j)).During to P16 (injecting back 14 days), cell has extensively infiltrated (Fig. 2 (k)) in the amphiblestroid capillary structure.Before putting to death animal during intravascular injection rhodamine-VISOSE (with identification function property retinal vessel), most Lin ^-The blood vessel aligning of HSC and stretching, extension (Fig. 2 (l)).The labeled cell of observing two kinds of patterns distributes: (l) in a pattern, cell scatters along the blood vessel between the unlabelled endotheliocyte; (2) another pattern demonstration blood vessel is made up of labeled cell fully.Institute's injected cells also penetrates in the blood vessel of dark plexus vasculosus (Fig. 2 (m)).Though once reported Lin in the past ^-HSC deutero-EPC sporadicly penetrates in the new vessel system, but this to be the reported first blood vessel network all be made up of these cells.This shows the bone marrow Lin of the present invention of intravitreal injection ^-Cell among the HSC crowd can effectively infiltrate in any layer of the retinal vessel clump in the formation.

When behind intravitreal injection, reaching 5 or 10 days whens inspection, the histological examination of non-retinal tissue (for example brain, liver, heart, lung, marrow) shows and does not have any GFP positive cell.This shows Lin ^-Cell subsets in the HSC part is target retina star spongiocyte optionally, and stable the infiltration in the developmental retinal vessel.Because these cells have many characteristics (with the association of retina star spongiocyte, protracted configuration, stablely infiltrate in the blood vessel that stretches and do not appear at the blood vessel external position) of endotheliocyte, so these cells show that EPC is present in Lin ^-Among the HSC crowd.By the star spongiocyte of target with observed in many hypoxemia property retinopathys be same type.As everyone knows, neurogliocyte be DR with the retina injury of other type in the main ingredient of viewed new vessel leaf.Under the situation of the neovascularization that active gliosis and local asphyxia cause; Active star spongiocyte hyperplasia, produce cytokine and raise GFAP, similar with observed situation in the newborn retinal vessel template forming process of the many mammalian species that comprise the people.

Lin of the present invention ^-Active star spongiocyte HSC crowd can resemble in newborn eye in the little rathole of target adult is with Lin ^-The HSC injection cell is gone into retina by in the adult eye of photocoagulation (Fig. 3 (a)) or needle point (Fig. 3 (b)) damage.In two models, be around damage location, to observe to have the painted cell mass of obvious GFAP (Fig. 3 (a and b)).From the Lin that is injected ^-The cell of HSC compsn is positioned at the damaged position, and keeps associate (Fig. 3 (a and b)) with the positive star spongiocyte of GFAP specifically.At these positions, also observe Lin ^-The HSC cell moves to amphiblestroid more deep layer, and its aspect of living in is similar with observed aspect during the newborn formation of deep layer retinal blood guard system.Amphiblestroid not damage location does not contain Lin ^-The HSC cell, this and Lin ^-When HSC is injected in the normally not impaired adult retina viewed consistent (Fig. 2 (e)).These data show, in the newborn retina of impaired adult retina with gliosis and experience vascularization, and Lin ^-The HSC compsn is the active neurogliocyte of target optionally.

The Lin of intravitreal injection ^-HSC can save and stable sex change vascular system.Because the Lin of intravitreal injection ^-HSC compsn target star spongiocyte also infiltrates in the normal retinal blood guard system, so these cells are also stablized the sex change blood vessel in local asphyxia relevant with gliosis and vascular degeneration or sex change retinal diseases.The rd/rd mouse is the retinal degeneration model, and it is to the degree of depth sex change of being born and showing sight sensor and retinal vessel layer back month the time.Retinal blood guard system normal development in these mouse is to P16, and this moment, darker plexus vasculosus was degenerated; Dark clump and the almost completely sex change of intermediate plexus of most of mouse during to P30.

In order to confirm whether HSC can save the blood vessel of degeneration, during P6 with Lin ⁺HSC or Lin ^-HSC (from the Balb/c mouse) intravitreal injection is gone in the rd/rd mouse.During to P33, injection Lin ⁺Behind the HSC, the blood vessel of the darkest layer of retina almost completely disappear (Fig. 4 (a and b)).Great majority have been injected Lin during on the contrary, to P33 ^-The retina of HSC has almost normal retinal blood guard system, and it has 3 vascular lamina (Fig. 4 (a and d) parallel, good moulding.The quantification of this effect shows, has injected Lin ^-The dark plexus vasculosus medium vessels mean length of rd/rd eye almost be to be untreated or Lin ⁺3 times (Fig. 4 (e)) of the eye that cell is handled.Surprisingly, injection derives from the Lin of rd/rd adult mouse (FVB/N) marrow ^-The HSC compsn has also been saved the rd/rd newborn mice retinal blood guard system (Fig. 4 (f)) of sex change.Just observe the sex change of rd/rd mouse eye medium vessels system 2-3 week as far back as the birth back.Injection Lin during late to P15 ^-HSC also can partially stabilized rd/rd mouse the sex change vascular system reach at least one month (Fig. 4 (g and h)).

Be injected to the Lin in children (for example P2) the rd/rd mouse in age ^-The HSC compsn also penetrates in the blood vessel of developmental top layer.During to P11, observe these cells and move to dark plexus vasculosus aspect, and form and viewed same pattern (Fig. 5 (a)) in the outside retinal vessel layer of wild-type.The Lin that injects for more clear description ^-Cell in the HSC compsn infiltrates and stablizes the mode of the sex change retinal blood guard system in the rd/rd mouse, with the Lin that derives from the Balb/c mouse ^-The HSC compsn is injected in the little rathole of Tie-2-GFP FVB.The FVB mouse has the rd/rd genotype, and owing to they expressed fusion protein Tie-2-GFP, so all endogenous blood vessels all fluoresce.

When with Lin ^-Unlabeled cells in the HSC compsn is injected into when also infiltrating in the developmental vascular system subsequently in the newborn Tie-2-GFP FVB eye; In the endogenous blood vessel of Tie-2-GFP mark the crack that is not labeled should be arranged, these cracks are corresponding to LinHSC injection, that infiltrate, that be not labeled.Use another kind of vascular markers (for example CD-31) dyeing subsequently, so just depict whole blood vessel, allow to confirm whether non-endogenous endotheliocyte is the part of vascular system.Inject latter two month, at injection Lin ^-Observe CD-31 male, the negative blood vessel (Fig. 5 (b)) of Tie-2-GFP in the eyes retina of HSC compsn.What is interesting is that most blood vessels of being saved contain Tie-2-GFP positive cell (Fig. 5 (c)).As that kind that smooth muscle actin dyeing is measured, no matter whether there is the blood vessel rescue, pericyte distributes not because of injection Lin ^-HSC and change (Fig. 5 (d)).These data clearly show, the Lin of the present invention of intravitreal injection ^-The HSC compsn moves in the retina in the hereditary defect mouse, participates in normal retinal vessel formation and stablizes the endogenous blood vessel in the sex change.

By from Lin ^-The transfectional cell of HSC suppresses retinal vessel to be taken place.Most retinal vascular disease relate to unusual blood vessel hyperplasia rather than sex change.The transgenic cell of target star spongiocyte can be used for transmitting angiogenesis inhibitor albumen and suppresses the blood vessel generation.With T2-tryptophanyl-tRNA synthetase (T2-TrpRS) transfection from Lin ^-The cell of HSC compsn.T2-TrpRS is the 43kD fragment (Fig. 6 (a)) that effectively suppresses the TrpRS of retinal vessel generation.The Lin of injection control plasmid transfection during P2 ^-The eyes retina of HSC compsn (no T2-TrpRS gene) has elementary normally (Fig. 6 (c)) and secondary (Fig. 6 (d)) retinal vessel clump when P12.As Lin of the present invention with the T2-TrpRS transfection ^-The HSC compsn is injected in the P2 eye and when postevaluation in 10 days, elementary network obviously unusual (Fig. 6 (e)), and the formation of dark retinal blood guard system almost completely is suppressed (Fig. 6 (f)).Observed minute quantity blood vessel attenuates in these eyes, has big gap between blood vessel.The Lin of secretion T2-TrpRS ^-The inhibition degree of HSC is specified in table 2.

T2-TrpRS is by Lin ^-Cell in the HSC compsn is in external generation and secretion, and these transfectional cells are injected into after the vitreum, in retina, observes the T2-TrpRS fragment (Fig. 6 (b)) of 30kD.This 30kD fragment is only at the Lin that injects transfection of the present invention ^-Could observe clearly in the retina of HSC, compare with reorganization or external synthetic albumen, the reduction of this apparent molecular weight maybe be owing to T2-TrpRS processing or degraded in vivo.These data show, Lin ^-The HSC compsn can be used for there being the active gene of function (for example expressing the gene that vasculogenesis suppresses molecule) to be passed to the retinal blood guard system through the active star spongiocyte of target.Due to the cell-mediated activity, this is extremely impossible though observed angiogenesis suppression action possibly be, because with the identical but Lin of non-T2 transfection ^-The eye of HSC combination treatment has normal retinal blood guard system.

Table 2.T2-TrpRS secretor type Lin ^-The blood vessel restraining effect of HSC

The Lin of intravitreal injection ^-HSC crowd is positioned to the retina star spongiocyte, infiltrates in the blood vessel and can be used for treating multiple retinal diseases.Though the injection the HSC compsn in most cells be attached to the star spongiocyte template, small amounts of cells in depth moves in the retina, go back to the nest to dark blood vessel network subsequently will be in the zone of this growth.Even if before back 42 days of birth, do not observe the positive star spongiocyte of GFAP, can not get rid of that the negative neurogliocyte of GFAP exists and be Lin in this zone ^-The HSC location provides the possibility of signal.Research has in the past shown that numerous disease is relevant with active gliosis.Especially in DR, neurogliocyte and extracellular matrix thereof take place relevant with pathologic vessels.

Because the Lin of injection ^-Be attached to the neurogliocyte of expressing GFAP, therefore no matter the damage of what type all can be used Lin of the present invention cell-specific in the HSC compsn ^-Damage location before blood vessel in the HSC compsn target retina takes place.For example, in the ischemic retinopathy such as mellitus, it is that anoxybiotic is reacted that new vessel forms.Through with Lin ^-HSC compsn target pathologic neovascularization position; Can stablize developmental new vessel system; Thereby prevent new vessel system exception (with the cause of disease of the relevant visual deprivation of DR), and can alleviate the anoxic that stimulates new vessel to form originally potentially such as hemorrhage or oedema.Abnormal vascular can be resumed into standard state.In addition, can use the Lin of transfection ^-The star spongiocyte activation of HSC compsn and induced with laser is delivered to the pathologic vessels happening part with vasculogenesis arrestin such as T2-TrpRS.Because the laser photocoagulation widespread usage is on Clinical Ophthalmology, this method has the application to multiple retinal diseases.Although in cancer therapy, studied these methods, because intraocular injection makes and might a large amount of cells directly be passed to disease sites, so these methods more have superiority to the purposes of eye illness based on cell.

Lin ^-The neurotrophic of HSC and the rescue of vascular nutrition property.Separate Lin with MACS by strengthening in green fluorescent protein (eGFP), CM (rd/rd), FVB (rd/rd) mouse bone marrow cells as stated ^-HSC.Will be from the Lin that contains EPC of these mouse ^-The HSC intravitreal injection is gone in P6C3H or the little rathole of FVB.Different time points after injection (1 month, 2 months and 6 months) is collected retina.Analyse vascular system through scan laser confocal microscope behind the CD31 antibody staining and the retinal tissue credit after the DAPI nuclear staining.Also use the microarray gene expression analysis of the retina mRNA of different time points to differentiate the gene that relates to this effect potentially.

The eyes of rd/rd mouse have the serious sex change of retina neural sensory layer and retinal blood guard system during to P21.When P6, use Lin ^-The rd/rd mouse eyes that HSC handles are kept normal retinal blood guard system and are reached 6 months; (1M, 2M and 6M) compares with contrast on all time points, deep layer and middle layer all be significantly improved (referring to Figure 12).In addition, we observe with respect to the Lin that uses as contrast ⁺The eye that HSC handles is used Lin ^-Also thicker (the 1M of retina that HSC handles; 1.2 doubly, 2M; 1.3 doubly, 6M; 1.4 doubly), have the more cell (1M of more number at outer nuclear layer; 2.2 doubly, 2M; 3.7 doubly, 6M; 5.7 doubly).(be untreated or non-Lin with contrast ^-Handle) the rd/rd retina compares, to " rescue " retina (for example, Lin ^-HSC) extensive genome analysis shows, the obvious rise of the gene of coding sHSP (little heat shock protein(HSP)) and the specificity growth factor relevant with neural rescue with blood vessel (comprising coding list in the figure A of Figure 20 and the proteic gene among the B).

Bone marrow Lin of the present invention ^-HSC crowd is remarkable and can repeatedly induce keeping of normal blood vessels system, and has greatly increased sight sensor and other neuronal cell layer in the rd/rd mouse.This neurotrophic rescue effect is relevant with the remarkable rise of little heat shock protein(HSP) and growth factor, the retinal degenerative disease that still can't cure is at present provided the thinking of treat-ment.

Rd1/rd1 mouse retina shows degree of depth blood vessel and neuronal degeneration.Normal birth back retinal vessel and the existing detailed description of neuronal development in the mouse, with viewed variation in the human foetus of third trimester pregnancy similar (Dorrell etc., 2002, Invest.Ophthalmol.Vis.Sci.43:3500-3510).Various features (the Frasson etc. of the mouse homozygote joint owner retinal degeneration of rd1 gene; 1999; Nat.Med.5:1183-1187), show sight sensor (PR) forfeiture fast, and with the serious blood vessel atrophy (Bowes etc. that cause by the sudden change in the PR cGMP phosphodiester enzyme coding gene; 1990, Nature347:677-680).Be vascular system in the inspection retinal development and sex change subsequently thereof; Use the antibody of the former protein I V of anticol (CIV) and the antibody (Figure 15) of anti-CD31 (PECAM-1); Extracellular matrix (ECM) albumen that the former protein I V of anticol is the mature blood guard system, CD31 is the endothelial cell marker thing.The retina normal development of rd1/rd1 (C3H/HeJ) until birth back (P) approximately 8 days, contains outer nuclear layer (ONL) the beginning sex change of sight sensor this moment.The quick sex change of ONL, cell is dead through apoptosis, only remaining individual layer nuclear when making to P20.The self-contained retina of antibody double staining with CIV and CD31 this two discloses, the vascular degeneration details in the rd1/rd1 mouse with other people described similar (Blanks etc., 1986, J.Comp.Neurol.254:543-553).Retinal vessel primary layer and deep layer seem to grow normal, and until P12, endotheliocyte loses fast afterwards, according to being CD31 dyeing disappearance.CD31 is positive, and endotheliocyte exists with proper distribution, until P12, but after this disappears fast.What is interesting is that the CIV positive staining all keeps existing at the full time point of being checked, prompting blood vessel and the ECM that links to each other normally form, but after P13, only keep matrix, do not observe the CD31 positive cell during to P13.(Figure 15, middle figure).The also sex change after P21 of middle plexus vasculosus, but progress will be slower than in dark clump viewed (Figure 15, last figure).For with rd1/rd1 mouse contrast, provided normal mouse retinal vessel and neurocyte layer (right figure, Figure 15).

Bone marrow Lin ^-The neuroprotective of HSC in the rd1/rd1 mouse.The Lin of intravitreal injection ^-HSC penetrates in the endogenous retinal blood guard system in whole three plexus vasculosuses, and prevents vascular degeneration.What is interesting is, in fact never observe injected cells at outer nuclear layer.These cells or penetrate in the retinal vessel in the formation, or near these blood vessels, be observed.Mouse Lin ^-Intravitreal injection is gone in the little rathole of C3H/HeJ (rd1/rd1) during the P6 of HSC (from C3H/HeJ) before sex change is about to begin.During to P30, injection control cells (CD31 ^-) the eye show typical rd1/rd1 phenotype, promptly in the retina of each inspection, all observe the deep-level blood vessel clump and the ONL of almost completely sex change.Injection Lin ^-The eye of HSC has kept normal apparent middle plexus vasculosus and dark plexus vasculosus.Surprisingly, at injection Lin ^-Observed cell is obviously more than the eye (Figure 16 (A)) of having injected control cells among the inner nuclear layer (INL) of the eye of HSC and the ONL.Lin ^-This rescue effect of HSC can be observed (Figure 16 (B)) in back 2 months in injection, and lasted up to 6 months (Figure 16 (C)).When relatively saved with do not saved at the moment, injected Lin ^-The intermediate plexus of the eye of HSC and dark clump vascular system and the INL and the difference among the ONL that contain neuronal cell are at all Measuring Time points all clearly (Figure 16 (B and C)).Through measuring vascular system total length (Figure 16 (D)) and counting that viewed DAPI positive cell check figure (Figure 16 (E)) quantizes this effect among the ONL.The data of all time points are used simple linear regression analysis.

At injection Lin ^-When P30 (p＜0.024) and P60 (p＜0.034), observe the statistics significant correlation property (Figure 16 (F)) between blood vessel rescue and neurone (the for example ONL thickness) rescue in the eye of HSC.When P180, relatively injected Lin ^-The retina of HSC when injecting the retina of control cells, although there is not significance,statistical (p＜0.14), dependency still very high (Figure 16 (F)).On the contrary, the retina of injecting control cells is put the significant correlation property (Figure 16 (F)) that does not all demonstrate between vascular system maintenance and the ONL at any time.These data show, intravitreal injection Lin ^-Retinal vessel and neurone that HSC causes occurring together in rd1/rd1 mouse retina are saved.Injected cells is not all observed in ONL or in the retinal vessel or any position of closing on beyond the retinal vessel.

Injected Lin ^-The amphiblestroid functional rescue of the rd/rd of HSC.To injection control cells or mouse Lin ^-The mouse of HSC latter two month is cooked electroretinogram ERG (ERG) (Figure 17).Immunohistochemical methods and microscopic analysis are carried out to every eye in ERG record back, to confirm that blood vessel and neurone rescue have taken place.From being shown by the representative ERG record of the treatment eye saved and the contrast eye of not saved, in the eye of being saved, the clear detectable signal (Figure 17) of the signal of reducing on the numeral (eye of treatment deducts untreated eye) the about 8-10 microvolt amplitude of generation.Obviously, from the signal severely subnormal of two eyes.Yet, by Lin ^-The eye of HSC treatment can record consistent and detectable ERG.In all examples, all be undetectable from the ERG that contrasts eye.Though the signal amplitude in the eye of being saved far below normal eyes, just can be observed signal as long as learn rescue in a organized way all the time, and signal amplitude is similar to signal based on other rescue research report of gene.All these results are illustrated in and use Lin of the present invention ^-There is functional rescue to a certain degree in the eye that HSC handles.

Rd/rd retina cell's type of being saved is mainly the cone.Use to retinal rod opsin or the specific antibody of cone opsin that saved with the immunohistochemical methods methods analyst with retina that do not saved.Same eye to being used for the ERG record that Figure 17 lists is carried out retinal rod opsin or the analysis of cone opsin.In the wild-type mice retina; The sight sensor less than 5% that exists is the cone (Soucy etc.; 1998; Neuron 21:481-493), the immunohistochemical staining collection of illustrative plates that is observed with red/green cone opsin or with the retinal rod Visual purple is shown in Figure 25 (A) or Figure 25 (B), and this collection of illustrative plates meets this percentage of cone cell.When the wild-type retina dyeed with preparatory immune IgG, dyeing (Figure 25 (C)) was not all observed in the optional position in the retina neural sensory layer beyond the blood vessel autofluorescence.Be born back 2 months, the retina of the rd/rd mouse of injection does not have the outer nuclear layer of basic atrophy, and it does not demonstrate any dyeing with red green cone opsin antibody (Figure 25 (D)) or Visual purple antibody (Figure 25 (G)).Injection contrast CD31 ^-The eye of HSC is not positive dyeing to the cone opsin (Figure 25 (E)) that exists or retinal rod opsin (Figure 25 (H)) yet.On the contrary, injection Lin ^-HSC has about 3 to about 8 row's nuclears to branch hole in maintained outer nuclear layer; The major part of these cells is to cone opsin positive (Figure 25 (F)), and about 1-3% is to retinal rod opsin positive (Figure 25 (I)).Remarkably be, this is with observed almost opposite by the normal mouse retina at first, and the normal mouse retina is main with retinal rod.These data show, injection Lin ^-HSC can keep the cone in the prolonging period of the general complaisance of the cone.

People's marrow (hBM) source Lin ^-HSC also saves the sex change retina.Separation is from the Lin of people's marrow ^-HSC shows to such an extent that be similar to mouse Lin ^-HSC.Collect marrow from people's donor, remove Lin ⁺HSC produces people Lin ^-HSC (hLin ^-HSC) crowd.These cells are with optical dye mark in vitro, and are injected in the little rathole of C3SnSmn.CB17-Prkdc SCID.The Lin of injection ^-HSC migrates to and target retinal vessel happening part, mode and injection mouse Lin ^-Observed mode identical (Figure 18 (A)) during HSC.Except blood-vessels target, people Lin ^-HSC also provides strong rescue effect (Figure 18 (B and C)) to the blood vessel of rd1/rd1 mouse and neurocyte layer this two.This observations confirms in people's marrow, to exist target retinal blood guard system also can prevent the cell of retinal degeneration.

Lin ^-HSC has vascular nutrition property and neurotrophic effect in the rd10/rd10 mouse.Although the rd1/rd1 mouse is to use the retinal degeneration model (Chang etc., 2002, Vision Res.42:517-525) the most extensive, that sign is the most detailed, sex change is very fast, and is different with observed slower usually time-histories in human diseases in this.In this strain, the photoreceptor cell sex change begins about P8, and P8 is that the retinal blood guard system is still in the moment (Figure 15) of stretching fast.Sex change takes place in deep layer retinal blood guard system subsequently, even if intermediate plexus is still when forming, therefore, the retina of rd1/rd1 mouse is from incomplete development, and this is with suffer from this sick philtrum at great majority viewed different.The rd10 mouse model has slower sex change time-histories, more closely is similar to the human retina denatured state, uses this model research Lin ^-The blood vessel rescue of HSC mediation.In the rd10 mouse, the photoreceptor cell sex change begins in about P21, just begins vascular degeneration thereafter soon.

Because accomplish substantially when normal retina neural sensory layer is grown to P21, so after retina thoroughly breaks up, begin to observe sex change, therefore, this sex change more is similar to the human retina sex change than rd1/rd1 mouse model.Will be from the Lin of rd10 mouse ^-HSC or control cells are injected in the P6 eye, to estimate retina on the time point that changes.When P21, injected Lin ^-The retina of the eye of HSC and control cells seems all normal, all growths fully of vascular lamina, INL and ONL normal development (Figure 18 (D and H)).Retinal degeneration begins to take place and aggravation in time when about P21.During to P30, the retina of injection control cells shows serious blood vessel and neuronal degeneration (Figure 18 (I)), and injection Lin ^-The retina of HSC keeps almost normal vascular lamina and photoreceptor cell (Figure 18 (E)).Difference between that saved and the eye do not saved on the time point of back more significantly (contrast Figure 18 (F and G) and Figure 18 (J and K)).In the eye of control treatment, through the immunohistochemical staining of CD31 and collagen protein IV most clearly being observed development (Figure 18 ((I-K)) of vascular degeneration.The eye of control treatment is almost completely negative to CD31, and collagen protein IV positive vessels " vestige " is still obvious, and what show generation is that blood vessel is degenerated, rather than uncompleted vascularization.On the contrary, Lin ^-The eye of HSC treatment has the positive and collagen protein IV male blood vessel of CD31 simultaneously, seems to be similar to very much normal wild-type eye (contrast Figure 18 (F and I)).

Use Lin ^-The amphiblestroid gene expression analysis of HSC treatment back rd/rd mouse.Use extensive genomics (microarray analysis) to analyze saved and retina that do not saved, to differentiate the mediators of inferring in the neurotrophic rescue.To use Lin ^-Genetic expression in the rd1/rd1 mouse retina of HSC treatment and the retina of not injection and injection control cells (CD31 ^-) retina compare.These are relatively respectively with triplicate enforcement.In three all duplicate samples, the expression level that requires gene to have is at least 2 times high of background level just can assert its existence.Compare with the rd/rd mouse retina of having injected control cells and not injection, at Lin ^-The gene that is raised 3 times in the retina of HSC protection is shown in figure A and B among Figure 20.Through with the average expression level of standard deviation divided by each cRNA replica, the variation coefficient of calculation expression gene (COV) level.In addition, through the association between related MV and standard deviation (SD) calculation expression level and the noise variance.Obtain the gene expression dose of each gene and the association between the standard deviation, make and to measure background level and credible expression level threshold value.Generally speaking, data drop in the tolerance range (Tu etc., 2002, Proc.Natl.Acad.Sci.USA 99:14031-14036) fully.Below the expression level that has of the gene discussed separately be higher than these critical expression levels.Paired " t check " value of the gene of discussing is also listed in table 1.Under various situation, the p value is rational (being near or below 0.05), shows to have similarity between the replica, and existing between the different test group maybe significant difference.The gene of many remarkable rises, comprise MAD and Ying Yang-1 (YY-1) (Austen etc., 1997, Curr.Top.Microbiol.Immunol.224:123-130), to avoid apoptosis relevant with the protection cell for the function that its proteins encoded had.Many crystallins with relate to the known heat shock protein(HSP) that the protection cell avoids stress and have sequence homology and similar function, these crystallins are also by Lin ^-HSC treats rise.The proteic expression of alpha-crystal is positioned at ONL (Figure 19) through immunohistochemical analysis.Figure 19 shows, uses Lin ^-HSC treatment back is being raised by crystallin α A in the outer nuclear layer cell of saving, but handle with control cells to branch hole in raise.Left figure has shown by the dyeing of the IgG in the retina of saving (contrast).Middle figure has shown by the crystallin α A in the retina of saving.Right figure has shown the crystallin α A in the retina of not saved.

Will be from personnel selection Lin ^-The amphiblestroid messenger RNA(mRNA) of rd1/rd1 mouse of HSC rescue is hybridized to human specific Affymetrix U133A micro-array chip.After strict the analysis, found many genes, its mRNA express be human specific, be higher than background, with mouse Lin ^-The retina of HSC rescue with injected retina people's control cells, that do not saved and compared, at people Lin ^-Obviously higher in the retina of HSC rescue (Figure 20, figure C).CD6 (a kind of at primary cell adhesion molecule of expressing with CD34+ hemopoietic stem cell surface new differentiation) and interferon alpha 13 (gene that another is expressed by hemopoietic stem cell) are all found through the microarray bioinformatics technique, have verified the validity of evaluation method.In addition, at people Lin ^-In the mouse retina sample of HSC rescue, several kinds of growth factors and neurotrophic factor are higher than background and express (Figure 20, figure D).

Use the negative bone marrow Lin that selects to contain EPC of lineage committed hematopoietic cell mark ^-HSC crowd.Though can be used as the bone marrow Lin of EPC ^-The HSC subgroup is not to be characterized by cell surface marker commonly used, but these cells in growth or the behavior in the impaired retinal blood guard system be different from Lin fully ⁺Or it is viewed among the mature endothelial cell crowd.These cell selective ground target retinal vessel happening parts, the formation of participating in stretching blood vessel.

Occur together the usually retinal vessel loss of hereditary retinal dystrophy disease.Effective treatment to this type disease requires functional rehabilitation and keeps complex organization's structure.Although nearest several researchs after deliberation the purposes of transmitting based on cell of nutritional factor or stem cell self, this two certain combination possibly be essential.For example, use factor therapy treatment retinal degenerative disease to cause the non-adjustable hypertrophy that saves land of blood vessel, cause the havoc of normal retinal tissue structure.Use nerve or retinal stem cells treatment retinal degenerative disease can rebuild neuronal function, but functional blood vessel also is essential to keeping the retinal function integrity.Lin of the present invention ^-The cell of HSC penetrates into rd/rd mouse retinal vessel, under the situation of destroying retinal structure not, stablizes the vascular system of sex change., injection cell also observes this rescue effect when going in the P15rd/rd mouse.Because the vascular degeneration in the rd/rd mouse is when P16, so this observations has been expanded effective Lin ^-The treatment window of HSC treatment.Injecting Lin of the present invention ^-Retinal neurons and sight sensor are able to keep in the eye of HSC, and sight function is maintained.

Adult bone marrow Lin ^-When HSC goes into to suffer from the mouse of retinal degenerative disease at intravitreal injection, apply the vascular nutrition property and the neurotrophic effect of the degree of depth.This rescue continues to reach 6 months after acting on treatment, and (be born back 16 days up to mouse, said mouse showed complete retinal degeneration usually in back 30 days to being born) injected Lin before complete retinal degeneration ^-The most effective during HSC.This rescue acts in two kinds of retinal degeneration mouse models and observes; And remarkably be;, acceptor uses adult's bone marrow HSC when being the immunodeficient rodent (for example SCID mouse) that suffers from retinal degeneration; Perhaps when donor be when suffering from the mouse of retinal degeneration, can realize this rescue.Though nearest several report has been described after the virus type gene rescue that utilizes wild type gene, suffer from the rescue of the mouse of retinal degeneration or the part phenotype in the dog (Ali etc., 2000, Nat Genet 25:306-310; Takahashi etc., 1999, J.Virol.73:7812-7816; Acland etc., 2001, Nat.Genet.28:92-95),, the present invention passes through the general cellular type therapeutic action that the blood vessel rescue obtains but being first.Therefore, it is more practical than the individual gene therapy of creating to each known mutations that this method has the potential use of one group of disease (for example retinitis pigmentosa) of known relevant sudden change more than 100 to treatment.

Neurotrophic rescue effect cutting subbase plinth really is not clear, but only under the situation that has the blood vessel that occurs together to stablize/save, observes.The existence of injection stem cell itself is not enough to produce neurotrophic rescue, and the obvious stem cell source neurone that do not exist has been got rid of the possibility that the cell of injecting changes sight sensor in outer nuclear layer.The data that obtained by the microarray gene expression analysis show that known gene with anti-apoptosis effect obviously raises.Because observed most of neuronal deaths all are the apoptosis mode in retinal degeneration, so this protection possibly have great curative effect with the key neuronic life-span of other sight function in these diseases, prolonging sight sensor.C-myc is that a kind of passing through raised various downstream apoptosis inducing factor and the transcription factor of apoptosis involvement.C-myc is expressed in to increase in the rd/rd mouse and is higher than 4.5 times of wild-types, shows that it possibly relate to observed sight sensor sex change in the rd1/rd1 mouse.Known two at Lin ^-It is active that gene M ad1 that significantly raises in the retina of HSC protection and YY-1 (Figure 20, figure A) suppress c-myc, thereby suppress c-myc inductive apoptosis.Show that also Mad1 crosses the activation of expression inhibiting Fas inductive Caspase-8, Caspase-the 8th, the another kind of key ingredient of apoptosis pathway.The rise of these two kinds of molecules can start through the apoptosis that prevents generally in the rd/rd mouse, to cause sex change and avoid playing a role aspect blood vessel and the neurodegeneration at the protection retina.

Another group is at Lin ^-The gene that significantly raises in the retina of HSC protection comprises crystallin family member (Figure 20, figure B).With heat shock protein(HSP) and other stress-induced protein similar, crystallin can stress be activated by retina, and the provide protection of anti-apoptosis is provided.In retina nutritional trouble rat model, the unusual low expression of α A-crystallin is relevant with the sight sensor loss, and a nearest amphiblestroid proteome analysis of rd/rd mouse shows that inducing that crystallin raises is the response to retinal degeneration.According to our amphiblestroid microarray data of rd/rd mouse of EPC rescue, the rise of crystallin seems in the retina neural protection of EPC mediation, to play an important role.

Gene and crystallin such as c-myc, Mad1, Yx-1 possibly be the downstream mediators of neurone rescue.Neurotrophic material adjustable anti-apoptotic genes expression is expressed, but we do not confirm to induce the known neurotrophic factor of increase level with the amphiblestroid microarray analysis of mouse stem cells rescue.On the other hand, show really that the expression of a plurality of growth factor genes is low but increase significantly with the rescue of human specific chip analysis people marrow derived stem cell mediation.

Up-regulated gene comprises some member and the Otoferlin of fibroblast growth family.Sudden change in the Otoferlin gene is relevant with genetic block, causes the deafness due to the auditory nerve disease.Might be Lin by injection ^-The Otoferlin that HSC produces also helps to prevent retinal nerve diseases.In history, suppose all the time that in the patient of retinal degeneration and animal it is because the secondary result that the dead caused metabolic demand of sight sensor reduces that observed blood vessel changes.This paper data show, concerning the mouse that suffers from hereditary retinal dystrophy, keep normal vascular system possibly also help to keep the component of outer nuclear layer at least.Recently the report in the document can be supported such viewpoint: the Nutrition that the tissue specificity vascular system has has exceeded the predictive role that blood vessel " nutrient " only is provided.For example, after the VEGFR1 activation, liver endothelial cell can be induced to produce liver cell regeneration and the key growth factor of keeping (LeCouter etc., 2003, Science 299:890-893) when meeting with liver injury.

It is relevant to have reported that indicative interaction similar between the hepatic parenchymal cells of vascular endothelial cell and vicinity and liver organ form, and for a long time just forms functional blood vessel afterwards.Endogenous retinal blood guard system in the retinal degeneration individuality maybe not can be facilitated so strong rescue; If but this vascular system supports with the endothelial progenitor cells that derives from the marrow hemopoietic stem cells crowd; Then they can make vascular system that sex change is more had resistance, help the survival of retinal neurons and blood vessel simultaneously.Suffering from the philtrum of retinal degeneration, postponing the extra visual performance that complete retinal degeneration outbreak can provide the several years.Use Lin of the present invention ^-The animal of HSC treatment has obviously kept supporting the ERG of its eyesight.

Recognize extensively that clinically although still can keep sight function, sight sensor and other neurone possibly have physical loss.In some cases, crucial threshold value intersects, visual deprivation.Because nearly all people's hereditary retinal dystrophy is all fallen ill early but morbidity is slow, can differentiate that therefore retinal degeneration is individual, and with ABM stem cell graft vitreum internal therapy of the present invention, to postpone retinal degeneration and the visual deprivation that occurs together.Be target and the infiltration that strengthens these stem cells, and the star spongiocyte of expectation existence activation (Otani etc., 2002, Nat.Med.8:1004-1010); This can realize by early treatment when having relevant gliosis, or realize by the active star spongiocyte of use laser stimulation local multiplication.Randomly, within the eye the injection before with one or more neurotrophy materials in vitro the transfection stem cell can be used for strengthening the rescue effect.This method can be used for treating other optic nerve unit degenerative disease, for example wherein has the glaucoma of retinal ganglion cells degeneration.

Lin of the present invention ^-HSC crowd is contained EPC crowd, its can be under the situation of destroying retinal structure not through the active star spongiocyte of target and infiltrate and promotes the blood vessel generation in the existing template.Lin of the present invention ^-HSC crowd also provides surprising long-term neurotrophic rescue effect in suffering from the eye of retinal degeneration.In addition, genetic modification, contain EPC from body Lin ^-HSC compsn portable is gone in the eye of local ischemic or abnormal vascularization, and can stablize and infiltrate in neovascularity and the neurone layer, and localized delivery is treated molecule constantly for a long time.This gene with significant dosage localized delivery expression medicine on the physiology has been represented a kind of new example of treating the eye illness that can't cure at present.

For example, the Photoreceptors in the normal mouse retina is mainly retinal rod, but is using Lin of the present invention ^-The observed outer nuclear layer in HSC rescue back mainly contains the cone.The sex change of most of heredity human retina it is generally acknowledged that by due to the primary retinal rod specific defects cone forfeiture is the secondary result of retinal rod functional disorder, and the disappearance of some nutritional factor that the retinal rod functional disorder possibly expressed with retinal rod is relevant.In the face of retinal rod/retinal degeneration the time by Lin ^-The promoted the inventive method of cone survival of inducing of HSC provides a method, is master's human macula lutea in the disease of for example retinitis pigmentosa, to keep the cone better.

Numerous variations and the modification in the above-mentioned embodiment be can implement and the spirit and the scope of new feature of the present invention do not deviated from.The specific embodiments that this paper sets forth unrestrictedly is intended to or should not be inferred to be restriction.

Claims

1. the purposes of hemopoietic stem cell crowd Mammals marrow source, isolating, lineage negative in the preparation medicine; Said medicine is used for improving the cone cell sex change of Mammals retina; Said hemopoietic stem cell crowd comprises hemopoietic stem cell and endothelial progenitor cells; Present in an amount at least sufficient to postpone the cone cell sex change in the retina, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 20% cell expressing surface antigen CD31.

2. the purposes of claim 1, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 50% cell expressing surface antigen CD31.

3. the purposes of claim 1, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 75% cell expressing surface antigen CD31.

4. the purposes of claim 1, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about the surface antigen of 50% cell expressing beta 2 integrin alpha 6.

5. the purposes of claim 1, wherein said hemopoietic stem cell crowd isolating, lineage negative derives from adult marrow.

6. the purposes of claim 1, wherein said hemopoietic stem cell crowd isolating, lineage negative comprises the mouse cell.

7. the purposes of claim 6, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 50% cell expressing surface antigen CD31, and at least about 50% cell expressing surface antigen CD117.

8. the purposes of claim 6, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 65% cell expressing surface antigen CD117.

9. the purposes of claim 6, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 80% cell expressing surface antigen CD31, and at least about 70% cell expressing surface antigen CD117.

10. the purposes of claim 1, wherein said hemopoietic stem cell crowd isolating, lineage negative comprises people's cell.

It is negative that 11. the purposes of claim 10, the cell among the hemopoietic stem cell crowd of wherein said isolating, lineage negative are CD133, the surface antigen of cell expressing beta 2 integrin alpha 6 at least about 50%, and at least about 50% cell expressing surface antigen CD31.

12. the purposes of claim 10, the cell among the hemopoietic stem cell crowd of wherein said isolating, lineage negative is the CD133 male, is less than the surface antigen of about 30% cell expressing beta 2 integrin alpha 6, and is less than about 30% cell expressing surface antigen CD31.

13. the purposes of claim 1 wherein by isolating hematopoietic stem cells crowd in the Mammals that suffers from eye illness, gives retina with said cell afterwards.

14. the purposes of claim 13, the hemopoietic stem cell crowd of wherein said lineage negative separates as follows:

(a) extract marrow by Mammals to be treated;

(b) from said marrow, separate multiple monocyte;

(c) with the said monocyte of pedigree colony antibody labeling to one or more antigenic biotin-conjugated in pedigree surface, wherein said pedigree surface antigen is selected from CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR and CD235a; With

(d) from multiple monocyte, remove monocyte, and reclaim the hemopoietic stem cell crowd of the lineage negative that contains endothelial progenitor cells said one or more pedigree surface antigen positives.

15. the purposes of claim 14, wherein said Mammals are mouse.

16. the purposes of claim 14, wherein said Mammals are mouse, said monocyte in step (c) with the pedigree colony antibody labeling of the biotin-conjugated of anti-CD3, CD11, CD45, Ly-6G and TER-119.

17. the purposes of claim 14, wherein said Mammals is behaved.

18. the purposes of claim 14; Wherein said Mammals is behaved, said monocyte in step (c) with the pedigree colony antibody labeling of the biotin-conjugated of anti-CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 and CD235a.

19. the purposes of claim 17, wherein said Mammals is behaved, with the CD133 antibody labeling monocyte of biotin-conjugated, and the hemopoietic stem cell crowd who reclaims CD133 male, lineage negative.

20. the purposes of claim 17, wherein said Mammals is behaved, and with the CD133 antibody labeling monocyte of biotin-conjugated, removes the CD133 positive cell, and reclaims CD133 hemopoietic stem cell crowd negative, lineage negative.

21. the purposes of claim 1, wherein said hemopoietic stem cell crowd isolating, lineage negative gives through intraocular injection.

22. the purposes of claim 21, wherein said disease are retinal degenerative disease.

23. the purposes of claim 21, wherein said disease are the ischemic retinopathy.

24. the purposes of claim 21, wherein said disease are angiorrbagia.

25. the purposes of claim 21, wherein said disease are vascular leakage.

26. the purposes of claim 21, wherein said disease are choroidopathy.

27. the purposes of claim 21, wherein said disease are AMD.

28. the purposes of claim 21, wherein said disease are diabetic retinopathy.

29. the purposes of claim 21, wherein said disease is for intending ocular histoplasmosis.

30. the purposes of claim 21, wherein said Mammals are newborn Mammals.

31. the purposes of claim 30, wherein said disease are retinopathy of prematurity.

32. the purposes of claim 21, wherein said disease are sicklemia.

33. the purposes of claim 21, wherein said disease are retinitis pigmentosa.

34. the purposes of claim 1, the hemopoietic stem cell crowd of wherein said isolating, lineage negative treats the gene transfection of useful peptide with efficient coding, gives the Mammals retina with said stem cell then.

35. the purposes of claim 34, the useful peptide of wherein said treatment is the angiogenesis inhibitor peptide.

36. the purposes of claim 34, wherein said angiogenesis inhibitor peptide is a protein fragments.

37. the purposes of claim 36, wherein said protein fragments are the angiogenesis inhibitor fragment of TrpRS.

38. the purposes of claim 37, the fragment of wherein said TrpRS are T2-TrpRS.

39. the purposes of claim 34, the useful peptide of wherein said treatment is the neurotrophic material.

40. the purposes of claim 39, wherein said neurotrophic material is selected from: NGFF, neurotrophin-3, neurotrophin-4, neurotrophin-5, CNTF, retinal pigment epithelium deutero-neurotrophic factor, rhIGF-1, glial cell-line deutero-neurotrophic factor and brain derived neurotrophic factor.

41. the purposes of claim 34, hemopoietic stem cell crowd wherein said transfection, lineage negative is prepared as follows:

(a) extract marrow by the adult Mammals;

(b) from said marrow, separate multiple monocyte;

(c) usefulness is to the said multiple monocyte of pedigree colony antibody labeling of the biotin-conjugated of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR and CD235a;

(d) from said multiple monocyte, separate monocyte to said one or more pedigree surface antigen positives, and the hemopoietic stem cell crowd who reclaims the lineage negative that contains endothelial progenitor cells; With

(e) treat the hemopoietic stem cell of the lineage negative that reclaims in the polynucleotide transfection step (d) of useful peptide with efficient coding.

42. by the isolating purposes of hemopoietic stem cell crowd in the preparation medicine that comprises the lineage negative of endothelial progenitor cells of Mammals marrow; Said medicine is used for keeping suffering from the cone cell of the Mammals retina of eye illness; Wherein isolating stem cell goes in the Mammals eye with the quantity intravitreal injection that is enough to improve the cone cell sex change in the retina, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 20% cell expressing surface antigen CD31.

43. the purposes of claim 42, wherein said disease are retinal degenerative disease.

44. the purposes of claim 42, the hemopoietic stem cell crowd of wherein said isolating, lineage negative treats the gene transfection of useful peptide with efficient coding, gives the Mammals retina with said stem cell then.

45. by the isolating purposes of hemopoietic stem cell crowd in the preparation medicine that comprises the lineage negative of endothelial progenitor cells of Mammals marrow; Said medicine is used for keeping suffering from the cone cell of the Mammals retina of eye illness; Wherein with the local multiplication of laser therapy retina with the activation star spongiocyte in the stimulation retina; Subsequently isolating stem cell is gone in the Mammals eye with the quantity intravitreal injection that is enough to improve the cone cell sex change in the retina, wherein in the hemopoietic stem cell crowd of said isolating, lineage negative at least about 20% cell expressing surface antigen CD31.