CN103290051B - Method for transdifferentiation of human fibroblast to retinal pigment epithelium - Google Patents
Method for transdifferentiation of human fibroblast to retinal pigment epithelium Download PDFInfo
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Abstract
The invention relates to a method for transdifferentiation of human fibroblast to retinal pigment epithelium. The method comprises the steps of transferring various transcriptional regulation factors and reporter genes into mammal fibroblast, and then, culturing the fibroblast with various culturing mediums to obtain the retinal pigment epithelium.
Description
Technical field
The present invention relates to cytodifferentiation field.Particularly, the present invention relates to the method that is retinal pigment epithelium by Mammals inoblast transdifferentiation in vitro.
Background technology
Retina degenerative disease is the degenerative lesion of special cells group in retina, often causes inpairment of vision even blind.Its main pathological basis is retina neuronic structure and function sexual abnormalities at different levels, finally causes the non-reversibility infringement of patient's vision.Macular degeneration is a kind of common retina degenerative disease, and blinding is high, mainly with retinal pigment epithelium dysfunction and aging relevant.Because the retinal degeneration disease of these blindings all exists progressive retina cell to lose, conventional pharmacological agent lacks curative effect, so desirable methods for the treatment of is to be replaced the cell of loss or played the effect of sustenticular cell by Transplanted cells, this feasible prevention of blindness method is applied to clinical more and more.The regenerative medicine treatment of retinal pigment epithelium is current clinical treatment retina degenerative disease means as the most effective in macular degeneration.
The source of Transplanted cells material is one of bottleneck of regenerative medicine treatment widespread use.Cell derived is in the past mainly to realize the differentiation from embryonic stem cell or inductive pluripotent stem cells to retinal pigment epithelium by following several technology: 1. utilize Spontaneous Differentiation method to obtain the technology (Meyer of retinal pigment epithelium from human pluripotent stem cell, J.S., et al.Modeling early retinal development with human embryonic and induced pluripotent stem cells.PNAS, 2009,106:16698-16703); 2. utilize individual layer differentiation method to obtain the technology (Nakano of retinal pigment epithelium from human pluripotent stem cell, T., et al.Self-formation of optic cups and storable stratified neural retina from human ESCs.Cell stem cell, 2012,10:771-785).3. utilize three-dimensional culture method to obtain the technology (Zhu of retinal pigment epithelium from human pluripotent stem cell, Y., et al.Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.PloS one, 2013,8, e54552).But embryonic stem cell can not be widely used in clinical treatment owing to being subject to ethical restriction, and although inductive pluripotent stem cells has been avoided ethical restriction, but its differentiation efficiency is very low, length consuming time, and may there is defect and the tumorigenicity of genetics and epigenetics, all limit its clinical application in Transplanted cells.
The appearance of transdifferentiation means makes the noble cells of a type be transformed into the noble cells of another kind of type, and this is just for the clinical application of Transplanted cells provides a large amount of cell deriveds.The stem cell stage has been walked around in conversion between this direct cell type, can make in theory the tumorigenesis risk of transplanting greatly reduce.Transdifferentiation technology can produce various kinds of cell type at present, as neurone, liver cell, epithelial cell etc.But also do not report at present to the transdifferentiation method of retinal pigment epithelium from human fibroblasts or other somatocyte type.The technology of the present invention makes clinical patient's autotransplantation material that can obtain in a large number the retina degenerative diseases such as treatment macular degeneration, and can be used for the treatment of the screening of retina degenerative disease medicine.
Summary of the invention
Directly from Mammals (for example the present invention seeks to, people) inoblast transdifferentiation is retinal pigment epithelium, for the clinical Transplanted cells material that the retina degenerative diseases such as a large amount for the treatment of macular degeneration are provided, and be applied to the screening for the treatment of retina degenerative disease medicine.
The present invention relates to a kind of method that is retinal pigment epithelium by Mammals inoblast transdifferentiation in vitro, described method comprises multiple transcriptional regulator and reporter gene is proceeded in Mammals inoblast, obtains retinal pigment epithelium thereby then use multiple substratum to cultivate.
More specifically, the invention provides the following:
1. a method that is retinal pigment epithelium by Mammals inoblast transdifferentiation in vitro, said method comprising the steps of:
A) structure contains respectively the transfection carrier of the gene order of encoding mammalian transcriptional regulator cMyc, MitfA, Otx2, Rax and Crx;
B) build the transfection carrier that contains the reporter gene under the control of retinal pigment epithelium specificity promoter;
C) by described step a) in build transfection carrier and described step b) in build transfection carrier be transfected in the described Mammals inoblast of cultivating for the substratum of cultivating mammalian somatic cell;
D) after transfection, change substratum for cultivating mammalian stem cell into and continue to cultivate until observe cell and start to occur that form changes for cultivating the substratum of mammalian somatic cell described;
E) by described for cultivate the substratum of mammalian embryonic stem cell change into contain matrigel continue to cultivate so that described Mammals inoblast further breaks up to the substratum of neural stem cell differentiation for mammalian embryonic stem cell;
F) the described substratum changing into for cultivating retinal pigment epithelium to the substratum of neural stem cell differentiation for mammalian embryonic stem cell that contains matrigel is continued to cultivate until obtain the positive cell clone of described reporter gene.
2. according to the method described in the 1st, wherein said Mammals is selected from people, rat, mouse, dog, cat, ox, rabbit, horse, pig, monkey etc.
3. according to the method described in the 2nd, wherein said Mammals is people.
4. according to the method described in the 1st, wherein said step a) also comprises that structure contains respectively the transfection carrier of the gene order of transcriptional regulator Pax6, Nrl and Klf4.
5. according to the method described in the 1st, described method also comprises successively cultivates the positive cell clone of described reporter gene until observe obvious melanocyte in the substratum continuation for cultivating retinal pigment epithelium that contains vitamin A acid (RA) and Sonic Hedgehog (SHH).
6. according to the method described in the 1st, wherein said retinal pigment epithelium specificity promoter is the promotor of Bestrophin1 (Best1) gene.
7. according to the method described in the 5th, wherein said Bestrophin1(Best1) sequence of the promotor of gene is as shown in SEQ ID NO.1 or SEQ ID NO.2
8. according to the method described in the 1st, wherein said reporter gene is the gene of fluorescin, the gene of for example green fluorescent protein.
9. according to the method described in the 1st, wherein said step a) and the carrier using b) be retroviral vector.
10. according to the method described in the 1st, the gene order of wherein said regulatory factor Rax is as shown in SEQ ID NO.3; The gene order of described regulatory factor Crx is as shown in SEQ ID NO.4; The gene order of described regulatory factor MitfA is as shown in SEQ ID NO.6; The gene order of described regulatory factor Otx2 is as shown in SEQ ID NO.7; The gene order of described regulatory factor cMyc is as shown in SEQ ID NO.9.
11. according to the method described in the 3rd, and the gene order of wherein said regulatory factor Pax6 is as shown in SEQ ID NO.5; The gene order of described regulatory factor Nrl is as shown in SEQ ID NO.8; The gene order of described regulatory factor Klf4 is as shown in SEQ ID NO.10.
12. according to the method described in the 1st, and wherein said is mammalian somatic cell substratum for cultivating the substratum of mammalian somatic cell; Described is CDF12 substratum for cultivating the substratum of mammalian embryonic stem cell; Described is N2/B27 substratum for mammalian embryonic stem cell to the substratum of neural stem cell differentiation; And described is RPE basic medium for cultivating the substratum of retinal pigment epithelium.
13. utilize the retinal pigment epithelium obtaining according to the method described in aforementioned any one.
Brief description of the drawings
Fig. 1: overall technological scheme of the present invention.
Fig. 2: the foundation of mankind RPE cell-specific Reporter System.
Fig. 3: hESC is to the qualification of Best::GFP positive cell in RPE cell differentiation procedure.
Fig. 4: the human fibroblast of transcriptional regulator mediation is to the strategy of RPE cell transdifferentiation.
Fig. 5: eight kinds of transcriptional regulator induction human fibroblasts are divided into Best1::GFP positive cell clone.
Fig. 6: the qualification of the Best1::GFP positive cell being come by human fibroblast's transdifferentiation.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Materials and methods:
1. substratum:
Mammalian somatic cell culture medium prescription:
DMEM/F12(Invitrogen，11320-033)
0.1mM non-essential amino acid (Invitrogen, 11140-050)
1mM GlutaMAX
tMdipeptides (Invitrogen, 35050-061)
1% penicillin/streptomycin (Invitrogen, 15070-063)
10% foetal calf serum (Hyclone, SH30084.83)
CDF12 culture medium prescription:
DMEM/F12(Invitrogen，11320-033)
0.1mM non-essential amino acid (Invitrogen, 11140-050)
1mM GlutaMAX
tMdipeptides (Invitrogen, 35050-061)
20%Knockout serum substitute (Invitrogen, N10828-028)
1% penicillin/streptomycin (Invitrogen, 15070-063)
55 μ M beta-mercaptoethanols (Invitrogen, 21985-023)
10ng/ml?Human?FGF2(Joint?Protein?Central)
N2/B27 culture medium prescription:
DMEM/F12(Invitrogen，11320-033)
0.1mM non-essential amino acid (Invitrogen, 11140-050)
1mM GlutaMAX
tMdipeptides (Invitrogen, 35050-061)
1% penicillin/streptomycin (Invitrogen, 15070-063)
1%N-2 additive (Invitrogen, 17502-048)
2%
additive (Invitrogen, 0080085-SA)
RPE basic medium:
DMEM/F12(Invitrogen，11320-033)
0.1mM non-essential amino acid (Invitrogen, 11140-050)
1mM GlutaMAX
tMdipeptides (Invitrogen, 35050-061)
1% penicillin/streptomycin (Invitrogen, 15070-063)
1%N-2 additive (Invitrogen, 17502-048)
5%Knockout serum substitute (Invitrogen, N10828-028)
Activin A substratum:
DMEM/F12(Invitrogen，11320-033)
0.1mM non-essential amino acid (Invitrogen, 11140-050)
1mM GlutaMAX
tMdipeptides (Invitrogen, 35050-061)
1% penicillin/streptomycin (Invitrogen, 15070-063)
20%Knockout serum substitute (Invitrogen, N10828-028)
100nM Activin A (R & D System, article No.: 338-AC)
2. hESC cultivates:
The human embryonic stem cell line H9 the present invention relates to obtains from U.S. WiCell Research Institute (article No.: WA09 (H9)-DL-7), utilizes two kinds of methods to cultivate:
A. H9 cell is seeded to and has cultivated in advance through mitomycin (purchased from Sigma company of the U.S., article No.: M0503) mouse embryo fibroblasts of deactivation is (purchased from American I nvitrogen company, article No.: S1520-100) culture plate in, end user's embryonic stem cell-like substratum (CDF12 substratum) and mouse embryo fibroblasts co-cultivation;
B. H9 cell is seeded to and uses in advance extracellular matrix (qualified-Matrigel, purchased from U.S. BD Biosciences, article No.: 354277) in coated culture plate, use mTeSR substratum (purchased from U.S. StemCell Technologies) to cultivate.
3. hESC is to the differentiation of retinal pigment epithelium:
HESC is digested with agglomerate form and be inoculated in the coated culture plate of 1% extracellular matrix, adding CDF12 cultivates based under 37 degrees Celsius and 5% carbon dioxide conditions and cultivates 1 hour, then use instead and contain 2%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: after N2/B27 substratum incubated overnight 354277), replace with again without qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: N2/B27 substratum 354277) continues to cultivate, within every two days, change a subculture, cell is divided into two groups afterwards, carry out follow-up cultivation according to following method respectively:
A.RA and SHH treatment group: from the 7th day of above-mentioned cultivation, to substratum, add 500nM vitamin A acid (RA, Sigma company of the U.S., article No.: R2625), vitamin A acid concentration reduces gradually afterwards, until the 200nM of the 10th day.The 11st to 14 days, substratum replaced with the N2/B27 substratum that contains 25nM Sonic Hedgehog (SHH, Sigma company of the U.S., article No.: SRP3156).Substratum is replaced by basic RPE substratum from the 15th day and continues to cultivate, above substratum is every two days and changes once.
B.Acvitin A treatment group: after above-mentioned cultivation the 10th to 20 days, substratum replaces with Activin A substratum, changes a subculture for every two days.Replacing with afterwards RPE basic medium and continue to cultivate, is within every two days, to change a subculture equally.
4. the structure of lentiviral vectors and retroviral vector:
Lentiviral vectors packaging plasmid is that pMDL, pCMV-VSVG and pRSV-REV (all buy article No.: 12251,35616 and 12253) from Addgene company of the U.S..Retroviral vector packaging plasmid be pCMV-GAG-Pol and pCMV-VSVG (purchased from Addgene company of the U.S., article No.: 14887 and 35616).The skeleton plasmid that builds reporter gene in Fig. 2-A is pGreenZeo lentiviral vectors (System Biosciences company of the U.S., article No.: #SR500VA/PA).In Fig. 4, be that pMX-gateway carrier is (purchased from Addgene company of the U.S., article No.: 18656) for the skeleton plasmid of inoblast transdifferentiation.Virus wrapping process is to utilize Lipofectamine2000 (purchased from American I nvitrogen company, article No.: 11668019) plamid vector transfection is entered to human embryonic kidney cell 293T (purchased from U.S. ATCC, article No.: CRL-11268), after 48 and 72 hours, collect culture supernatant, and filter with 0.45 μ mPVDF film (purchased from Millipore company of the U.S., article No.: SLHV033).
5. be Best1::GFP positive cell by human fibroblast's transdifferentiation:
First primary mankind inoblast (HFF) is incubated at and adds 1% penicillin/streptomycin (Invitrogen, in mammalian somatic cell substratum 15070-063), within every two days, change a subculture, can carry out virus infection until Growth of Cells density reaches 50-70%.Virus infection the day before yesterday, substratum is replaced by not containing 1% penicillin/streptomycin (Invitrogen, MEF substratum 15070-063), with every hole 75, the density of 000 cells/well, be inoculated into and use in advance 0.5%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: in 6 orifice plates of 354277) processing, under 37 degrees Celsius and 5% carbonic acid gas culture condition, cultivate 12 hours, directly in substratum, add afterwards the virus vector mixture of mentioning in specific embodiment 2, comprise eight kinds of retrovirus and a kind of lentiviral vectors, cells infected after 12 hours is replaced by substratum CDF12 substratum.Change afterwards liquid every day, after virus infection 5-6 days, can observe cellular form and start to become circle (as shown in Fig. 5-A) from fusiformis.After virus infection the 7th day, substratum is replaced by the N2/B27 substratum that contains 2%qualified-Matrigel, continue to cultivate after 8-24 hour, then substratum is replaced by RPE basic medium.Within every two days afterwards, change a subculture, after 20 days, under fluorescent microscope, select the BEST1::GFP positive cell clone that can send green fluorescence through blue light illumination at virus infection, be seeded to and use in advance 0.5%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: in 24 orifice plates of 354277) processing, first by the N2/B27 culture medium culturing of adding vitamin A acid (RA) and SHH, within every two days, change a subculture.Cultivate and within 10 days, afterwards substratum is replaced by RPE basic medium (changing a subculture for every two days), continue to cultivate until can observe obvious melanocyte.
The primer sequence using during 6.qPCR detects:
Embodiment 1.
1. be based upon Reporter System specific expressed in human retinal pigment epithelial cells.
Show owing to having been reported, mankind Bestrophin1(Best1) promotor of gene and Rpe65 gene, can in the retinal pigment epithelium (RPE) of transgenic mice, drive specifically the high efficient expression of reporter gene, therefore we are first by the mankind Bestrophin1(Best1 of different sizes) the promoter fragment clone of gene and Rpe65 gene enters lentiviral gene group carrier pGreenZeo (System Biosciences company of the U.S., article No.: #SR500VA/PA) upstream site (as shown in Fig. 2-A) of Green fluorescence protein gene (gfp), comprising 4 kinds of Best1 gene promoters, (length is respectively 0.2kb, 0.3kb, 0.6kb and 1kb) and 2 kinds of Rpe65 gene promoters (length is respectively 0.8kb and 1kb) (as shown in Fig. 2-A and table 1), then utilize lentiviral vectors packaging plasmid pMDLg/pRRE, pCMV-VSVG and pRSV-REV (all buy from Addgene company of the U.S., article No.: 12251, 35616 and 12253) carry out lentiviral vectors packaging.Concrete viral wrapping process is: utilize Lipofectamine2000 (purchased from American I nvitrogen company, article No.: 11668019) lentiviral gene group carrier and packaging plasmid cotransfection are entered to human embryonic kidney cell 293T (ATCC, CRL-11268), after 48 and 72 hours, collect culture supernatant, and filter with 0.45 μ m pvdf membrane (purchased from Millipore company of the U.S., article No.: SLHV033).Utilize the different lentiviral vectorss that build to infect multiple human cell, comprise the primary inoblast (HFF of the mankind, purchased from German Lonza company, article No.: CC-2509), human embryos nephrocyte 293T is (purchased from U.S. ATCC, article No.: CRL-11268), hESC H9 is (purchased from U.S. WiCell Research Institure, article No.: WA09 (H9)-DL-7) and the primary RPE cell of the mankind (purchased from German Lonza company, article No.: 194987) etc.Result of study shows, only has Best1-0.6kb (0.6kb, sequence is as shown in SEQ ID NO.1) and Best1-1kb (1kb, sequence is as shown in SEQ ID NO.2) two kinds of promotors can drive the high efficient expression of green fluorescent protein (as shown in Fig. 2-A) specifically in the primary RPE cell of the mankind, therefore we adopt Best1-0.6kb promotor wherein to carry out follow-up research, and constructed Reporter System is hereinafter also referred to as Best1::GFP Reporter System.
The checking of the Reporter System (Best1::GFP) of 2.RPE cell specific expression.
Method 1: in order further to verify the specificity of Best1::GFP reporter gene, first we infect hESC H9 (purchased from U.S. WiCell Research Institure with the lentiviral vectors of expressing Best1::GFP reporter gene (Best1-0.6kb promotor), article No.: WA09 (H9)-DL-7), then utilize the method (Meyer having reported, J.S., et al.Modeling early retinal development with human embryonic and induced pluripotent stem cells.PNAS, 2009, 106:16698-16703.) be the mixed cellularity group including RPE cell by H9 cytodifferentiation, result of study shows that Best1::GFP reporter gene only expresses in the RPE like cell with typical sexangle form, and in other neuron or fibroblast-like noble cells, be showed no expression (as shown in Fig. 2-B).
Method 2: first infect hESC H9 (purchased from U.S. WiCell Research Institure with the lentiviral vectors of expressing Best1::GFP reporter gene (Best1-0.6kb promotor), article No.: WA09 (H9)-DL-7), and 10 clones of picking, (test kit is purchased from Qiagen company of the U.S. for its genomic dna of extracting respectively, article No.: 10243), verify that by PCR method it is Best1::GFP reporter gene positive colony, then positive colony is continued to amplification cultivation and utilize the method (Boucherie having reported, C., et al.Brief report:self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors.Stem Cells, 2013, 31:408-414, Zhu, Y., et al.Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.PloS one, 2013, 8, e54552.) to RPE cell directional differentiation (as shown in Activin A group in Fig. 3-A and B), after 3 weeks, observe the monolayer cell of expressing pigment, simultaneously the experimental result of immunofluorescence shows that these cells can both express MITF, the RPE such as Best1 and ZO-1 cell typical marks thing (as shown in Fig. 3-E), prove that we are successfully divided into hESC RPE cell.In addition we,, also by two kinds of signal factors relevant with pigmented to RPE maturation of interpolation RA and SHH of novelty, have successfully strengthened the pigmented level (as shown in RA+SHH group in Fig. 3-A and B) of the RPE cell of differentiation.
On the other hand, from breaking up 21 days, we started to observe the cell of the Best1::GFP reporter gene positive in Activin A group and RA+SHH group, and positive rate can be increased to approximately 50% (as shown in Fig. 3-C) after breaking up 40 days, in noble cells, Best1mRNA level also significantly raises (as shown in Fig. 3-D) after starting to break up 3 weeks simultaneously, illustrates that the expression of Best1::GFP reporter gene can directly reflect the dynamic process of RPE cytodifferentiation visually.In addition we utilize qPCR method to detect to break up the eye early development gene (as shown in Fig. 3-F) in Best1::GFP reporter gene positive cell after 40 days, find that two kinds of key gene Mitf that RPE differentiation is relevant and the expression of Otx2 obviously raise, and other mature RPE cells specific gene, as Best1, Peaf, Rpe65, Cralbp, Tyr and Tyr2, also all there is high expression level, and the expression pattern of these genes is very similar at Best1::GFP reporter gene positive cell and the primary RPE cell of the mankind, further prove that we have successfully set up the Reporter System Best1::GFP of mankind RPE cell-specific.
Embodiment 2. human fibroblasts are to the transdifferentiation of retinal pigment epithelium.
We utilize PCR method to obtain six kinds to break up relevant transcriptional regulator to retinal development and RPE and (comprise Rax (NM_013435.2, sequence is as shown in SEQ ID NO.3), Crx (NM_000554.2, sequence is as shown in SEQ ID NO.4), Pax6 (NM_000280.2, sequence is as shown in SEQ ID NO.5), MitfA (NM_198159.2, sequence is as shown in SEQ ID NO.6), Otx2 (NM_021728.2, sequence is as shown in SEQ ID NO.7) and Nrl (NM_006177.2, sequence is as shown in SEQ ID NO.8)) complete encoding sequence of the transcriptional regulator (comprising cMyc (sequence is as shown in SEQ ID NO.9) and Klf4 (sequence is as shown in SEQ ID NO.10)) of (as shown in Fig. 4-A) and two kinds of high expression levels in mature RPE cells cell, and utilize conventional molecular biology method (with reference to " molecular cloning " third edition, Pehanorm Brooker, Science Press, 2008) its clone is entered to pMX-gateway carrier (purchased from Addgene company of the U.S., article No.: 18656) thus built the reverse transcription virus gene group carrier of expressing the different factors, concrete grammar is: utilize the primer of 8 kinds of different transcriptional regulator full length sequences of amplification and the PrimeStar archaeal dna polymerase (article No.: DR010B) of Japanese TaKaRa company, obtain the DNA fragmentation of 8 kinds of different transcriptional regulator complete encoding sequences by PCR method, because hold at its 5 ' end and 3 ' restriction enzyme site that adds respectively restriction enzyme EcoR I (5 '-GAATTC-3 ') and Xho I (5 '-CTCGAG-3 ') when design of amplification primers, so 8 kinds of different transcriptional regulator full length DNA fragments that obtain and pMX-gateway carrier are used after the restriction enzyme EcoR I (article No.: R0101) of NEB company of the U.S. and Xho I (article No.: R0146) carry out double digestion simultaneously, utilize the QIAquick Gel extraction kit of Qiagen company of the U.S. (article No.: 28706) glue reclaims endonuclease bamhi, finally utilize the T4DNA ligase enzyme (article No.: M0202) of NEB company of the U.S. that 8 kinds of different transcriptional regulator full length DNA fragments are connected and entered in pMX-gateway carrier, obtain the reverse transcription virus gene group carrier of expressing 8 kinds of different transcriptional regulators.Then utilize Lipofectamine2000 (purchased from American I nvitrogen company, article No.: 11668019) respectively by different reverse transcription virus gene group carriers and retroviral vector packaging plasmid pCMV-GAG-Pol and pCMV-VSVG (purchased from Addgene company of the U.S., article No.: 14887 and 35616) common transfection enters human embryonic kidney cell 293T (purchased from U.S. ATCC, article No.: CRL-11268), after 48 and 72 hours, collect culture supernatant, and with 0.45 μ m pvdf membrane (purchased from Millipore company of the U.S., article No.: SLHV033) filter, thereby build the retroviral vector of expressing respectively 8 kinds of factors.Further, we mix the lentiviral vectors equimolar ratio example of expressing the expression Best1::GFP reporter gene (promotor is wherein Best1-0.6kb promotor) building in eight kinds of transcriptional regulator retroviral vectors and embodiment 1 to make virus mixture, then utilize this virus mixture to infect primary inoblasts of the mankind (HFF) (purchased from German Lonza company, article No.: CC-2509) realize the transdifferentiation from human fibroblast to RPE cell, detailed process is as shown in Fig. 4-B: first HFF is incubated at and adds 1% penicillin/streptomycin (Invitrogen, mammalian somatic cell substratum 15070-063) (can with replacing for other substratum of cultivating mammalian somatic cell), within every two days, change a subculture, until reaching 50-70%, Growth of Cells density can carry out virus infection.Virus infection the day before yesterday, substratum is replaced by not containing 1% penicillin/streptomycin (Invitrogen, MEF training liquid 15070-063), with every hole 75, the density of 000 cells/well, be inoculated into and use in advance 0.5%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: in 6 orifice plates of 354277) processing, under 37 degrees Celsius and 5% carbonic acid gas culture condition, cultivate 12 hours, directly in substratum, add afterwards above-mentioned virus mixture, after cells infected 4-24 hour, substratum is replaced by CDF12 substratum (can replace with other substratum for cultivating mammalian embryonic stem cell), change afterwards liquid every day and once (discard whole original substratum, add fresh CDF12 substratum), after virus infection 5-6 days, can observe cell and start to occur that form change (becomes circle from fusiformis, as shown in Fig. 5-A).After virus infection the 7th day, substratum is replaced by and contains 0.5-2%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: N2/B27 substratum (can replace to other substratum of neural stem cell differentiation for embryonic stem cell) 354277), continue to cultivate after 8-24 hour, then substratum is replaced by RPE basic medium (can with replacing for other substratum of cultivating RPE cell).Within every two days afterwards, change a subculture and (discard whole original substratum, add fresh RPE basic medium), after the 21st day, under fluorescent microscope, select the BEST1::GFP positive cell clone that can send green fluorescence through blue light illumination at virus infection, be seeded to and use in advance 0.5-2%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: in 24 orifice plates of 354277) processing, with successively adding vitamin A acid (RA, Sigma company of the U.S., article No.: R2625) and Sonic Hedgehog (SHH, Sigma company of the U.S., article No.: SRP3156) RPE base culture base: postvaccinal 7-10 days add RA, its working concentration reduces by sky, be followed successively by 500nM, 400nM, 300nM and 200nM (changing a subculture every day), within postvaccinal 11-14 days, add SHH, its working concentration is 25nM (changing a subculture for every two days), continue to cultivate until can observe obvious melanocyte (using in addition 100nM Activin A process to replace RA and SHH to process also can realize transdifferentiation is RPE cell, uses RA and SHH just can observe the RPE cell expressing melanochrome that transdifferentiation forms but only have).Express respectively eight kinds of transcription factors and mCherry reporter gene (red fluorescent protein gene by utilizing, be used to indicate virus transfection effect, its plamid vector construction is identical with eight kinds of transcription factors with virus packaging) the experiment of retroviral vector coinfection HFF, we find that the transfection efficiency of independent a kind of virus vector reaches approximately 77% (as shown in Fig. 4-D), and we infer that infected cell can express the probability of above-mentioned eight kinds of transcription factors simultaneously and can reach 12% accordingly.Virus vector mixture coinfection is collected all HFF after 3 days and is carried out qPCR analysis (the primer is shown in materials and methods part), the expression (as shown in Fig. 4-C) of all 8 kinds of transcriptional regulators can be detected.
Utilize after the lentiviral vectors coinfection HFF of the expression Best1::GFP reporter gene (promotor is wherein Best1-0.6kb promotor) building in the retroviral vector of eight kinds of different transcriptional regulators of above-mentioned expression and embodiment 1 carries out transdifferentiation 12 days, we have observed directly the Best1::GFP reporter gene positive cell clone that can send green fluorescence through blue light illumination by fluorescent microscope, transdifferentiation after 21 days these cell clones start to show active proliferation activity, transdifferentiation after 35 days cell clone sprawl into gradually monolayer cell state, and start to form the form (as shown in Fig. 5-A) of RPE precursor cell sample.
Further, we have set 8 experimental group, reject respectively a kind of transcription regulaton factor for every group, utilize the lentiviral vectors coinfection HFF that expresses the retroviral vector of seven kinds of factors of residue and express Best1::GFP reporter gene (Best1-0.6kb promotor) to carry out transdifferentiation (as shown in Fig. 5-B), result shows to reject cMyc, after MitfA or Otx2, can't detect Best1::GFP reporter gene (Best1-0.6kb promotor) positive cell completely, in addition reject the quantity (as shown in Fig. 5-B) that Rax or Crx also can significantly reduce Best1::GFP reporter gene positive cell, prompting cMyc, MitfA, Otx2, Rax and Crx are for most important to the process of Best1::GFP reporter gene (Best1-0.6kb promotor) positive cell transdifferentiation by human fibroblast, and other three kinds of factor K lf4, Nrl and Pax6 can be rejected separately or be replaced by other factors.
The qualification of embodiment 3. transdifferentiation cells.
Using after infecting HFF cell the 21st day of virus mixture, under fluorescent microscope, select the BEST1::GFP positive cell clone that can send green fluorescence through blue light illumination, and be seeded to and use in advance 0.5%qualified-Matrigel (purchased from U.S. BD Biosciences, article No.: in 24 orifice plates of 354277) processing, cell is divided into two experimental group, first use and add 1 respectively) Activin A (final concentration 100nM, R & D System, article No.: 338-AC), or 2) RA (vitamin A acid, Sigma company of the U.S., article No.: R2625) and SHH (working concentration 25nM, Sigma company of the U.S., article No.: SRP3156) N2/B27 substratum, under 37 degrees Celsius and 5% carbon dioxide conditions, continue to cultivate, within every two days, change a subculture, to promote its further ripe (as shown in Fig. 6-B).Cultivate and within 10 days, afterwards substratum is replaced by RPE basic medium (changing a subculture for every two days), continue to cultivate until can observe obvious melanocyte.Using after infecting HFF cell the 35th day of virus mixture, the cell of two experimental group all shows and the similar cellular form of RPE cell being become by hESC's directed differentiation, uses real-time quantitative PCR (qPCR) method the expression (as shown in Fig. 6-A) of the RPE cell-specific molecular marked compounds such as MITF, ZO-1 and Best1 also can be detected simultaneously.The more important thing is, in the Best1::GFP positive cell of the culture medium culturing that now early application contains RA and SHH, can observe obvious melanocyte (as shown in Fig. 6-B), illustrate that this culture condition can make Best1::GFP positive cell more ripe and become the RPE cell with function.For further this discovery of checking, we use the Best1::GFP positive cell (adding the experimental group of RA and SHH) of flow cytometry enrichment maturation, and with the transcription product of RPE cell-specific sex factor in qPCR technical Analysis (the primer is shown in materials and methods part) cell, by with the primary inoblast comparison of the mankind, we find that ripe Best1::GFP positive cell not only expresses the transcriptional regulator manually proceeding to, but also the gene of expressing other intraocular enrichments such as Six3 and Lhx2, a series of direct and Vitamin A Metabolism can be detected in addition, the relevant genetic expressions of RPE function such as the synthetic and somatomedin secretion of melanochrome, as Best1, Cralbp, Pedf and Tyrp2 etc. (as shown in Fig. 6-C).
Claims (11)
1. a method that is retinal pigment epithelium by Mammals inoblast transdifferentiation in vitro, said method comprising the steps of:
A) structure contains respectively the transfection carrier of the gene order of encoding mammalian transcriptional regulator cMyc, MitfA, Otx2, Rax and Crx;
B) build the transfection carrier that contains the reporter gene under the control of retinal pigment epithelium specificity promoter, the sequence of described promotor is as shown in SEQ ID NO.1 or SEQ ID NO.2;
C) by described step a) in build transfection carrier and described step b) in build transfection carrier be transfected in the described Mammals inoblast of cultivating for the substratum of cultivating mammalian somatic cell;
D) after transfection, change substratum for cultivating mammalian embryonic stem cell into and continue to cultivate until observe cell and start to occur that form changes for cultivating the substratum of mammalian somatic cell described;
E) by described for cultivate the substratum of mammalian embryonic stem cell change into contain matrigel continue to cultivate so that described Mammals inoblast further breaks up to the substratum of neural stem cell differentiation for mammalian embryonic stem cell;
F) the described substratum changing into for cultivating retinal pigment epithelium to the substratum of neural stem cell differentiation for mammalian embryonic stem cell that contains matrigel is continued to cultivate until obtain the positive cell clone of described reporter gene.
2. method according to claim 1, wherein said Mammals is selected from people, rat, mouse, dog, cat, ox, rabbit, horse, pig, monkey.
3. method according to claim 2, wherein said Mammals is people.
4. method according to claim 1, wherein said step a) also comprises that structure contains respectively the transfection carrier of the gene order of transcriptional regulator Pax6, Nrl and Klf4.
5. method according to claim 1, described method also comprises successively cultivates the positive cell clone of described reporter gene until observe obvious melanocyte in the substratum continuation for cultivating retinal pigment epithelium that contains vitamin A acid (RA) and Sonic Hedgehog (SHH).
6. method according to claim 1, wherein said reporter gene is the gene of fluorescin.
7. method according to claim 1, wherein said reporter gene is the gene of green fluorescent protein.
8. method according to claim 1, the carrier that wherein said step is used in a) and b) is retroviral vector.
9. method according to claim 1, the gene order of wherein said regulatory factor Rax is as shown in SEQ ID NO.3; The gene order of described regulatory factor Crx is as shown in SEQ ID NO.4; The gene order of described regulatory factor MitfA is as shown in SEQ ID NO.6; The gene order of described regulatory factor Otx2 is as shown in SEQ ID NO.7; The gene order of described regulatory factor cMyc is as shown in SEQ ID NO.9.
10. method according to claim 4, the gene order of wherein said regulatory factor Pax6 is as shown in SEQ ID NO.5; The gene order of described regulatory factor Nrl is as shown in SEQ ID NO.8; The gene order of described regulatory factor Klf4 is as shown in SEQ ID NO.10.
11. method according to claim 1, wherein said is mammalian somatic cell substratum for cultivating the substratum of mammalian somatic cell; Described is CDF12 substratum for cultivating the substratum of mammalian embryonic stem cell; Described is N2/B27 substratum for mammalian embryonic stem cell to the substratum of neural stem cell differentiation; And described is RPE basic medium for cultivating the substratum of retinal pigment epithelium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102618488A (en) * | 2012-03-15 | 2012-08-01 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing retinal pigment epithelia |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102618488A (en) * | 2012-03-15 | 2012-08-01 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing retinal pigment epithelia |
Non-Patent Citations (4)
| Title |
|---|
| Direct conversion of human ibroblasts into retinal pigment epithelium-like cells by defined factors;Kejing Zhang 等;《Protein Cell》;20130620;第5卷(第1期);48-58 * |
| Kejing Zhang 等.Direct conversion of human ibroblasts into retinal pigment epithelium-like cells by defined factors.《Protein Cell》.2013,第5卷(第1期),48-58. * |
| 胡甘露 等.视网膜色素上皮细胞的诱导分化及其移植治疗方法的研究进展.《国际眼科纵览》.2010,第34卷(第5期),329-333. * |
| 视网膜色素上皮细胞的诱导分化及其移植治疗方法的研究进展;胡甘露 等;《国际眼科纵览》;20101031;第34卷(第5期);329-333 * |
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Inventor after: Liu Guanghui Inventor after: Zhang Kejing Inventor after: Ren Ruotong Inventor after: Li Ying Inventor after: Yi Fei Inventor after: Qu Jing Inventor after: Juan Carlos Izpisua Belmonte Inventor before: Liu Guanghui Inventor before: Zhang Kejing Inventor before: Ren Ruotong Inventor before: Li Ying Inventor before: Yi Fei Inventor before: Qu Jing |
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| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: LIU GUANGHUI ZHANG KEJING REN RUOTONG LI YING YI FEI QU JING TO: LIU GUANGHUI ZHANG KEJING REN RUOTONG LI YING YI FEI QU JING JUAN CARLOS IZPISUA BELMONTE |
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |