WO2021039972A1 - 尿路上皮細胞への誘導剤及び尿路上皮細胞の誘導方法 - Google Patents
尿路上皮細胞への誘導剤及び尿路上皮細胞の誘導方法 Download PDFInfo
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Definitions
- the present invention mainly relates to a method for inducing urothelial cells, in particular, a method for inducing urothelial cells by direct reprogramming, and an agent for converting somatic cells to urothelial cells.
- urothelial cells make up the urothelium.
- the urothelium contracts or dilates depending on the amount of urine stored and provides a strong barrier to urine exudation and bacterial invasion.
- urinary tract diseases such as bladder cancer, overactive bladder, congenital bladder abnormalities, bladder injury, interstitial cystitis, neurogenic bladder, atrophic bladder, urinary tract epithelial cell loss and / or dysfunction Is related.
- Non-Patent Document 1 A method of co-culturing human fetal bone marrow-derived mesenchymal stem cells with a patient's urothelial cells (Non-Patent Document 1) and a method of culturing human adipose-derived stem cells by adding a culture supernatant of urothelial cells (non-Patent Document 1).
- Patent Document 2 has been reported. However, these cells are rarely used clinically because the method requires autologous urothelial cells.
- iPS cells induced pluripotent stem cells
- the present invention provides a method for preparing urothelial cells that can be applied to the treatment of urothelial diseases, particularly diseases caused by damage to urothelial cells, diseases caused by loss or dysfunction of urothelial cells, and the like. It is an object of the present invention to provide urothelial cells prepared by the method and a medium for inducing urothelial cells suitable for the method.
- the present inventors have conducted intensive studies based on the findings so far. Specifically, we attempted to prepare urothelial cells from somatic cells without going through a pluripotent state by direct reprogramming technology. Direct reprogramming can be achieved by transducing genes encoding transcription factors that play important roles in the differentiation stage of target cells or genes that promote somatic cell reprogramming.
- the present inventors have found that human fibroblasts can be directly converted into urothelial cells by introducing several genes, establish the conversion procedure, and obtain the direct conversion.
- the phenotype and function of urothelial cells (dUC) were analyzed to complete the present invention. That is, the present invention is as follows.
- FOXA1 Formhead box A1 gene or its expression product
- TP63 tumor protein P63
- MYCL L-Myc gene or its expression product
- a method for inducing urothelial cells which comprises the step of introducing at least one selected from the group consisting of the KLF4 (Kruppel-like factor 4) gene or its expression product.
- KLF4 Kruppel-like factor 4
- the exogenous factor is selected from the following; (i) KLF4 gene or its expression product (ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product.
- the exogenous factor is selected from the following; (i) KLF4 gene or its expression product (ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product.
- exogenous factor is selected from the following; (i) KLF4 gene or its expression product (ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product.
- FOXA1 gene or its expression product As an exogenous factor FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and A vector for converting mammalian somatic cells into urothelial cells, which comprises at least one selected from the group consisting of the KLF4 gene or its expression product.
- the exogenous factor contains the FOXA1 gene or its expression product, and the TP63 gene or its expression product.
- exogenous factor is selected from the following; (i) KLF4 gene or its expression product (ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product.
- Urinary tract disease is at least one selected from the group consisting of bladder cancer, overactive bladder, congenital bladder abnormalities, bladder injury, interstitial cystitis, neurogenic bladder and atrophic bladder.
- the therapeutic agent according to the above [18].
- a medium for inducing urothelial cells which comprises 3-isobutyl 1-methylxanthine (IBMX) and epidermal growth factor (EGF).
- urothelial cells can be prepared from somatic cells in a short period of time by direct reprogramming. Since these urothelial cells can be easily derived from the somatic cells of the person to be transplanted, problems such as immunological rejection do not occur even when the obtained urothelial cells are transplanted. In addition, since urothelial cells can be directly induced from somatic cells without going through pluripotent states such as iPS cells and ES cells, problems caused by pluripotent stem cells such as canceration can be avoided.
- aHDFs were seeded on 12-well plates at a concentration of 3x10 4 cells / well and cultured in standard medium. The next day (day 0), the gene of interest was introduced with a retroviral vector. Unless otherwise specified, the cells were cultured in standard medium until the 4th day of introduction, and then changed to CnT-Prime medium and cultured once every 3-4 days while changing the medium. Real-time RT-PCR, phase-contrast microscopy, and immunocytochemical analysis were performed on day 21. It is a figure which shows the result of having investigated whether the fibroblast is converted into the urothelial cell by the gene transfer using the transcription factor associated with the urothelial cell.
- AHDFs were seeded on an uncoated 12-well plate and the FOXA1 (F), IRF1 (I), TP63 (T) and / or sonic hedgehog (SHH) (H) genes were introduced (bottom of A, black). The part indicates infection with each retroviral vector).
- the cells were cultured in standard medium for the period of day 1-3 and in CnT-Prime medium for the period of day 4-21.
- aHDFs were seeded on a laminin-coated 12-well plate and the F, I, T, H, POU5F1 (P), KLF4 (K) and / or MYCL (L) genes were introduced (bottom of figure, black to Gray areas indicate infection with the corresponding retroviral vectors).
- aHDFs The combination of several transcription factors promoted UPK1b expression in aHDFs. It is a figure which shows the result of having investigated whether the fibroblast is converted into the urothelial cell by the gene transfer using the transcription factor associated with the urothelial cell and the reprogramming gene in combination. Transfection of F, T, P, K, and L resulted in the formation of urothelial cell colonies expressing UPK1b.
- aHDFs were seeded on a laminin-coated 12-well plate and the gene group shown in the black to gray part at the bottom of (A) was introduced. The cells were cultured in standard medium for the period of day 1-3 and in CnT-Prime medium for the period of day 4-21.
- RNA was extracted from cells, and the UPK1b gene expression level was measured by real-time RT-PCR. The mRNA extracted from HUCs was measured in the same manner. The values shown by mean ⁇ SD were normalized by ⁇ -actin mRNA expression and were shown relative to the value of the non-transgenic control as 1.0 (N 3).
- B A phase-contrast microscopic image of a typical cell is shown. Shooting magnification: 20 times.
- FIPKL (FOXA1, IRF1, POU5F1, KLF4, MYCL), FTPKL (FOXA1, TP63, POU5F1, KLF4, MYCL), FHPKL (FOXA1, SHH, POU5F1, KLF4, MYCL) and FPKL (FOXA1, SHH, POU5F1, KLF4, MYCL) and FPKL
- aHDFs were seeded on a laminin-coated 12-well plate and the gene group shown in the black to gray part at the bottom of (A) was introduced.
- Cells were cultured in standard medium for the period 1-3 and CnT-Prime medium for the period 4-21.
- FTPKL (FOXA1, TP63, POU5F1, KLF4, MYCL), FTPK (FOXA1, TP63, POU5F1, KLF4), FTLK (FOXA1, TP63, MYCL, KLF4) and FTPL (FOXA1, TP63, POU5F1, MYCL).
- aHDFs were seeded on a 12-well plate coated with laminin. The next day (day 0), a retroviral vector containing any of the FOXA1 (F), TP63 (T), MYCL (L), and KLF4 (K) genes was introduced into cells. The introduced gene is indicated by "+”. Cells were cultured in standard medium for the period 1-3 and CnT-Prime medium for the period 4-21.
- B, C aHDFs were cultured on plates uncoated (N) or coated with collagen (C), poly-L-lysine (P), laminin (L). After introducing the F, T, L, and K genes with a retrovirus (day 0), the cells were cultured in the same manner as in (A).
- the dUCs in the figure are 21 days after gene transfer.
- A A typical phase-contrast microscopic image of the cells shown in the figure. The bar is 100 ⁇ m.
- each column shows the results of UPK1a, UPK1b, UPK2, UPK3a and UPK3b from the left side.
- RNA was extracted from aHDFs and iPS cells on the day shown in the figure. The expression of the gene shown in the figure was measured by real-time RT-PCR. Gene expression of HUCs was also measured. The values shown by mean ⁇ SD were normalized by ⁇ -actin mRNA expression and were shown relative to the value in iPS cells as 1.0 (N 3). N. S.
- the values shown by mean ⁇ SD were normalized by ⁇ -actin mRNA expression, and the values at aHDFs without gene transfer were shown relative to 1.0 (N 3).
- AHDFs introduced with F, T, L, and K were cultured in selective medium (P0) and subsequently subcultured (P1 to P4).
- A Conceptual diagrams of cell conversion and passage in the experiments performed in FIGS. 14-17.
- B, c Immunohistochemical staining image (b) of P0 dUCs and P4 dUCs stained with UPK1b, UPK2, E-cadherin (CDH1) and keratin 8/18 (KRT8 / 18), and HDFs, P0 dUCs. , UPK1b, UPK2, E-cadherin (CDH1) and keratin 8/18 (KRT8 / 18) -positive cells in dUCs of P4 (c).
- each column shows the results of UPK1b and UPK2 from the left side
- each column shows the results of UPK1a, UPK3a, UPK3b and E-cadherin (CDH1) from the left side.
- CDH1 E-cadherin
- KEGG pathway analysis diagram comparing dUCs and aHDFs. Genes that are highly expressed in dUCs are shown. It is a figure which shows the result of having investigated the distribution in the bladder tissue of the dUCs transplanted into the bladder.
- F, T, L, K and GFP were introduced into aHDFs with a retroviral vector and cultured in CnT-Prime medium for 21 days.
- the mRNA expression level of the illustrated gene was quantified by real-time RT-PCR (a), and representative phase-contrast microscope and fluorescence microscope images were shown (b).
- the epithelial tissue of the bladder was stained with the antibody shown in the figure (1st stage, UPK1b; 2nd stage, UPK2; 3rd stage, KRT8 / 18; 4th stage, CDH1; 5th stage, CDH1; 6th stage. Eyes, CDH1). Nuclei were stained with DAPI.
- M and L represent muscle and bladder lumens, respectively. Arrowheads indicate GFP-labeled dUCs. The bar is 100 ⁇ m.
- aHDFs were seeded on laminin-coated 12-well plates.
- day 0 a retroviral vector containing any or a combination of the FOXA1 (F), TP63 (T), MYCL (L), and KLF4 (K) genes was introduced into cells.
- the cells were cultured in standard medium for the period 1-3 and CnT-Prime medium for the period 4-14.
- RNA was extracted from cells and the mRNA levels of UPK1b and UPK2 were measured by real-time RT-PCR.
- the present invention relates to a method for inducing urothelial cells by converting differentiated somatic cells of mammals into urothelial cells.
- converting is meant converting somatic cells into the desired urothelial cells.
- One of the preferred embodiments of the method of the present invention is to urinate somatic cells without going through the process of cell initialization represented by the production of iPS cells, which is also called “direct reprogramming” or “direct conversion”. It is a method of converting to urothelial cells.
- Induction to urothelial cells can be performed either in vitro (in vitro) or in vivo (in vivo).
- Urothelial cells are cells that line the inside of the urethra from the renal cup, renal pelvis, through the ureter and bladder to the urethra, and in one embodiment, The urethral epithelial cells may be epithelial cells derived from the bladder.
- somatic cells to be directly reprogrammed are not particularly limited, and may be derived from mammals.
- somatic cells autologous cells
- urothelial cells prepared in advance from somatic cells of another person or another animal can be used for transplantation instead of autologous cells.
- urothelial cells can be produced from somatic cells of another person or another animal prepared in advance and used for transplantation. That is, a bank of urothelial cells can be prepared and used for transplantation purposes.
- MHC can be typed in advance in order to reduce the risk of rejection response and the like.
- the cell characteristics and tumorigenicity of urothelial cells can be confirmed in advance.
- mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cows, horses, pigs, and the like, and humans in particular.
- somatic cells somatic cells that can be easily collected from the living body can be used.
- fibroblasts keratinocytes, oral mucosal epithelial cells, nasal mucosal epithelial cells, airway mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, smooth muscle cells (bladder smooth muscle cells, vascular smooth muscle cells, etc.) ), Fat cells, gingival cells (gingival fibroblasts, gingival epithelial cells), dentin cells, root membrane cells, bone marrow cells, bone marrow-derived stromal cells, leukocytes, lymphocytes, conjunctival epithelial cells, osteoclasts, etc.
- fibroblasts Preferably fibroblasts, keratinocytes, oral mucosal epithelial cells, gingival cells, leukocytes, lymphocytes and the like. More preferably, it is a fibroblast. In the present invention, it is preferable to use the above-mentioned cells collected from a living body.
- the target somatic cells are preferably cells existing at a site where supply of urothelial cells is desired.
- the site where the supply of urothelial cells is desired is the inner surface of the bladder, and therefore the somatic cells as the starting material are the cells existing on the inner surface of the bladder. It is preferable to have.
- somatic cells used as starting materials include fibroblasts, bladder smooth muscle cells, vascular endothelial cells, vascular smooth muscle cells, and lymphocytes existing in the bladder.
- the FOXA1 Formhead box A1
- the TP63 tumor protein P63
- MYCL L-Myc
- At least one, preferably two, more preferably three, and even more preferably all four, consisting of the gene or its expression product and the KLF4 (Kruppel-like factor 4) gene or its expression product are introduced.
- the "expression product” include mRNA or protein of the FOXA1 gene, the TP63 gene, the MYCL gene and the KLF4 gene.
- one embodiment of a preferred combination of exogenous factors introduced into somatic cells comprises a combination of the FOXA1 gene or its expression product and the TP63 gene or its expression product.
- the method of the invention in which the exogenous factor is selected from the following is also a preferred embodiment.
- KLF4 gene or its expression product ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product. Both can enhance the expression of UPK2 mRNA.
- the TP63 gene or its expression product in addition to the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product, microRNA, siRNA, shRNA and DNA expressing them. Can also be used together. Moreover, various proteins can also be used together.
- genes that are highly conserved in vertebrates, and in this specification, unless a specific animal name is indicated, they represent genes including homologues.
- a gene having a mutation, including polymorphism, which has a function equivalent to that of a wild-type gene product is also included.
- nucleotide sequences of the human (Homo sapiens) FOXA1 gene, TP63 gene, MYCL gene and KLF4 gene and the amino acid sequences of the proteins encoded by them are provided by GenBank provided by the National Center for Biotechnology Information (NCBI).
- the method of the present invention can be carried out according to a known direct reprogramming method, except that a specific gene is selected, and can be carried out, for example, according to the method of any of the following documents: 1: Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors; Masaki Ieda, Ji-Dong Fu, Paul Delgado-Olguin, Vasanth Vedantham, Yohei Hayashi, Benoit G. Bruneau, and Deepak Srivastava Cell 142: 375-386, 2010. 2: Direct conversion of fibroblasts to functional neurons by defined factors. Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Kokubu, Thomas C.
- the gene of interest which is an exogenous factor
- the gene of interest which is an exogenous factor
- a method for introducing a gene in addition to a method of infecting a viral vector such as a retrovirus vector, an adenovirus vector, a lentivirus vector, an adeno-associated virus vector, a herpesvirus vector, or a Sendai virus vector, a gene and its expression product
- a viral vector is preferable from the viewpoint of transfer efficiency and stable retention of the transgene, and a plasmid is preferable to reduce the risk of canceration.
- a gene that serves as a drug selection marker puromycin resistance, blastsidedin S resistance, neomycin resistance, hygromycin resistance, etc.
- a gene that serves as a drug selection marker puromycin resistance, blastsidedin S resistance, neomycin resistance, hygromycin resistance, etc.
- the gene of the present invention may be introduced using a plasmid or a viral vector, for example, a retroviral vector.
- a viral vector is preferable from the viewpoint of transfer efficiency and stable retention of the transgene, and a plasmid is preferable to reduce the risk of canceration.
- the gene introduced into the somatic cell can be transcribed by the LTR promoter or expressed from another promoter inside the vector.
- Constitutive expression promoters such as the CMV promoter, EF-1 ⁇ promoter, CAG promoter, or the desired inducible promoter can be utilized.
- a chimeric promoter in which a part of the LTR is replaced with another promoter may be used.
- the transducing factor is a gene expression product (for example, protein)
- a peptide called Protein Transmission Domain (PTD) is bound to the expression product protein and added to the medium to introduce it into somatic cells. You may.
- differentiated somatic cells of mammals can be cultured in a medium after introduction of a gene.
- a medium for example, it is a preferred embodiment for inducing (preparing) urothelial cells in vitro.
- Culturing can be carried out in a suitable container for storing cells and medium.
- a suitable culturing method a method of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5% is exemplified, but the method is not limited thereto. Culturing under the above conditions can be carried out using, for example, a known CO 2 incubator.
- the period for culturing is not particularly limited as long as the effect of the present invention is not impaired. For example, it can be about 12 hours to 1 month, about 1 day to 3 weeks, and about 3 days to 2 weeks. If necessary, the medium can be changed.
- the culture conditions are preferably according to a conventional method.
- subculture In culturing, subculture can be performed if necessary. For passage, cells are harvested before or shortly after reaching confluence and seeded in fresh medium. In addition, in the culture of the present invention, the medium can be changed as appropriate.
- the medium used in the method of the present invention is not particularly limited. Ordinary liquid media such as MEM (Modified Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), and ⁇ MEM (alpha Modified Minimum Essential Medium) can be used. If necessary, serum components (Fetal Bovine Serum (FBS), Human Serum (HS)), antibacterial agents such as streptomycin and penicillin, and non-essential amino acids (NEAA) can be added. it can.
- MEM Modified Minimum Essential Medium
- DMEM Dynamic Eagle's Medium
- EMEM Eagle's minimal essential medium
- ⁇ MEM alpha Modified Minimum Essential Medium
- serum components Fetal Bovine Serum (FBS), Human Serum (HS)
- antibacterial agents such as streptomycin and penicillin
- NEAA non-essential amino acids
- a differentiation-inducing medium usually used for differentiating urothelial cells refers to a medium containing a component capable of differentiating pluripotent stem cells (ES cells, iPS cells, etc.) into urinary tract epithelial cells (hereinafter, Also referred to as the urinary tract epithelial cell induction medium of the present invention).
- the urothelial cell induction medium a known medium can be used.
- the medium described in 1 can be used. However, it is not limited to these.
- the urothelial cell induction medium of the present invention contains 3-isobutyl 1-methylxanthine (IBMX) and epidermal growth factor (EGF).
- IBMX 3-isobutyl 1-methylxanthine
- EGF epidermal growth factor
- the concentration of IBMX in the medium is usually 1 ⁇ M to 10 mM, but 1 mM to 10 mM IBMX is preferably contained in the medium for selective maintenance and proliferation of urothelial cells.
- the concentration of EGF in the medium is usually 0.001 to 1 ng / mL, preferably 0.01 to 1 ng / mL. The inclusion of these components makes the conversion of fibroblasts to urothelial cells and subsequent selective maintenance and proliferation of urothelial cells more efficient, as shown in the Examples.
- a preferred example of the urothelial cell-inducing medium of the present invention is a medium having the following composition (also referred to as “UCM medium” in the present specification).
- urothelial cells are derived from somatic cells.
- the formation of urothelial cells can be detected by, for example, gene expression of Uroplakin (Uroplakin1b; UPK1), Uroplakin2 (UPK2), CK5, CK17, formation of Asymmetric Unit Membrane, and the like.
- the induced urothelial cells are selected as exogenous factors from the group consisting of the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product. It has one type, preferably two types, more preferably three types, and even more preferably all four types.
- the exogenous factor includes the FOXA1 gene or its expression product, and the TP63 gene or its expression product, depending on the type and number of genes introduced. In yet another embodiment, it has the following genes or expression products thereof, or a combination thereof.
- KLF4 gene or its expression product ii) TP63 gene or its expression product, and KLF4 gene or its expression product (iii) MYCL gene or its expression product, and KLF4 gene or its expression product (iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (v) TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product (vi) FOXA1 gene or its expression product, TP63 gene or its expression product, and KLF4 gene or its expression product (vii) FOXA1 gene or its expression product, TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product.
- ign mainly refers to a mode of a gene or an expression product thereof introduced as a result of the above-mentioned introduction means, which is different from a natural mode.
- a gene whose expression is controlled by a promoter other than the natural promoter, a position on a non-natural chromosome, or an aspect of a gene existing outside the chromosome can be mentioned.
- the urothelial cells may be obtained as a mixture with cells other than the urothelial cells (for example, the original somatic cells). In such a case, urothelial cells and cells other than urothelial cells can be separated, if necessary.
- the means for separation is not particularly limited. For example, it can be separated using a cell sorter or magnetic beads.
- the urothelial cells induced by the present invention can be suitably used as, for example, a transplant material.
- the urothelial cells induced by the present invention can also be used for various studies and technological developments using urothelial cells. For example, it is useful for basic research such as analysis of the mechanism of urinary organ development, differentiation, and morphogenesis, and the effects of mechanical stress, nutrition, hormones, etc. on them.
- urinary tract epithelial cells can be easily, quickly, and inexpensively established from humans and animals having various diseases and genetic backgrounds, and thus are related to the diseases and genetic backgrounds. It is possible to analyze abnormalities of urinary tract epithelial cells by biochemical, molecular biological, immunological, etc. methods, which will be useful for research such as elucidation of the pathogenic mechanism of diseases and development of diagnostic methods. Can be done. Further, if such urothelial cells are used for drug development, drug toxicity test, etc., it can be useful for the development of new therapeutic methods for various diseases.
- the urothelial cells obtained by the present invention can be used for treating various diseases, particularly urinary system diseases.
- the urothelial cells induced by the present invention can be used in the formation of a substitute bladder in a case of total cystectomy for bladder cancer, the construction of a patch to the site where the urothelium is defective in a bladder-vaginal fistula, and the neuropathic bladder. Formation of urothelium to partially supplement urinary bladder enlargement when bladder enlargement is performed for cases where the bladder is fibrotic and atrophed It is useful for such purposes.
- Specific diseases to be treated include bladder cancer, overactive bladder, congenital abnormalities of the bladder (eg, bladder valgus, bladder diverticulum, abnormalities of the urinary tract), bladder damage, and interstitial bladder inflammation. (Hannah type and non-Hannah type), neurogenic bladder, atrophic bladder and the like can be mentioned.
- treatment is intended to be a treatment performed while a patient is suffering from a particular disease or disorder, thereby the severity of the disease or disorder, or one or more. It means that the multiple symptoms are alleviated or the progression of the disease or disorder is delayed or slowed down. In the present specification, “treatment” shall include “prevention”.
- the urothelial cells obtained by the present invention can also be used not only for the treatment of diseases but also for the purpose of cosmetology and functional enhancement.
- the treatment for humans is also referred to as treatment for convenience in the present specification, and "patient” can be read as "healthy person” or “human”, and “disease” can be read as “cosmetology” or "function”.
- the present invention can also be used for the treatment of diseases of not only humans but also pet animals such as dogs and cats and mammals including domestic animals such as cows, horses, pigs, sheep and chickens.
- pet animals such as dogs and cats and mammals including domestic animals such as cows, horses, pigs, sheep and chickens.
- "patient” shall be read as “patient” or "mammal”.
- composition As described above, at least one selected from the group consisting of the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product as an exogenous factor.
- Urothelial cells can be induced by introducing into somatic cells, preferably 2 types, more preferably 3 types, and even more preferably all 4 types. Therefore, the present invention further comprises at least one selected from the group consisting of the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product, preferably 2.
- compositions for inducing urothelial cells comprising species, more preferably three, and even more preferably all four.
- the composition for inducing the urinary tract epithelial cells contains a factor used for inducing the urinary tract epithelial cells from somatic cells, and is the FOXA1 gene or its expression product, the TP63 gene or its expression product, A form in which at least one, preferably two, more preferably three, and even more preferably all four selected from the group consisting of the MYCL gene or its expression product and the KLF4 gene or its expression product can be introduced into somatic cells. It is desirable that it is included in.
- the exogenous factor includes the FOXA1 gene or its expression product, and the TP63 gene or its expression product, depending on the type and number of genes introduced.
- the following genes or expression products thereof, or combinations thereof are included.
- KLF4 gene or its expression product ii) TP63 gene or its expression product, and KLF4 gene or its expression product
- MYCL gene or its expression product iii) MYCL gene or its expression product
- KLF4 gene or its expression product iv) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product
- TP63 gene or its expression product TP63 gene or its expression product, MYCL gene or its expression product
- KLF4 gene or its expression product ii) FOXA1 gene or its expression product, MYCL gene or its expression product, and KLF4 gene or its expression product
- FOXA1 gene or its expression product TP63 gene or its expression product, MYCL gene or its expression product, and KLF4 gene
- composition can be used, for example, as a drug (therapeutic agent) in gene therapy.
- Direct reprogramming in vivo Somatic cells present at the site of urinary epithelial cell defects include the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product.
- the FOXA1 gene or its expression product By introducing at least one, preferably two, more preferably three, and even more preferably all four selected from the group consisting of the expression product and the KLF4 gene or its expression product, on the urinary tract at the site of injury. It can induce cutaneous cells by direct reprogramming, thereby contributing to the treatment of urinary epithelial cells and the regeneration of urinary tract cells.
- the above composition of the present invention can be preferably used in the introduction of the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product.
- the induction method of the present invention includes direct reprogramming in vivo as described above in addition to direct reprogramming in vitro.
- gene therapy for treating various diseases can be performed, for example.
- Specific examples of the disease include those described above.
- somatic cells eg, fibroblasts
- somatic cells eg, fibroblasts
- somatic cells eg, fibroblasts
- the FOXA1 gene or its expression product e.g. fibroblasts
- the TP63 gene or its expression product e.g. TP63 gene or its expression product
- the MYCL gene or its expression e.g. TP63 gene or its expression product
- the MYCL gene or its expression e.g., fibroblasts
- At least one selected from the group consisting of the FOXA1 gene or its expression product, the TP63 gene or its expression product, the MYCL gene or its expression product, and the KLF4 gene or its expression product in a lumen such as in the bladder or renal pelvis can be done by injecting seeds, preferably two, more preferably three, and even more preferably all four. Inject the above gene or its expression product or a combination thereof into the bladder or renal pelvis with a urinary tract catheter or the like, and bring the somatic cells existing in the bladder or renal pelvis into contact with the gene or its expression product or a combination thereof. Induces cells to urothelial cells.
- ⁇ List of abbreviations >> aHDF (adult Human Dermal Fibroblast; adult human skin fibroblast) UC (urothelial cell) HUC (Human urothelial cell) HUVEC (Human Umbilical Vein Endothelial Cell) dUC (Direct Converted UC) FOXA1 (Forkhead box A1; also referred to as "F” in this specification) TP63 (tumor protein P63; also referred to as "T” in the present specification) MYCL (L-Myc; also referred to as "L” in the present specification) KLF4 (Kruppel-like factor 4; also referred to as "K” in the present specification) IRF1 (Interferon regulatory factor 1; also referred to as "I” in the present specification) GFP (green fluorescent protein)
- aHDF adult Human Dermal Fibroblast; adult human skin fibroblast
- UC urothelial cell
- HUC Human urothelial cell
- HUVEC Human Umbil
- the "Uromedium" medium contains 60 ⁇ M calcium (Gibco; MEP1500CA), 60 ⁇ g / mL bovine pituitary extract (Gibco), 5 ⁇ g / mL human recombinant insulin (Diagnocine), and 500 ng / mL hydrocortisone (500 ng / mL hydrocortisone).
- EpiLife TM medium was 60 ⁇ M calcium and 60 ⁇ g / mL bovine pituitary extract, 5 ⁇ g / mL human recombinant insulin, gentamicin amphotericin, 2% FBS, 0.01 ng / mL human recombinant EGF, 1 mM IBM X. It consists of EpiLife TM medium supplemented with 1 ⁇ M tranyl cypromin (Abcam).
- iPS cells were placed in StemFit medium (Ajinomoto; AK02N) on a plate coated with Easy iMatrix-511 silk (Nippi; 0.25 ⁇ g / cm 2; pre-treated at 37 ° C. for 1 hour). It was cultured. For passage, iPS cells were dissociated into a single cell suspension in StemFit medium containing a Rock inhibitor (Nacalai Tesque; Y-27632) and reseeded in new culture plates. The next day, the medium was replaced with a new StemFit medium containing no Rock inhibitor, and the medium was replaced every two days. Passage was performed when iPS cells reached 80-90% confluence.
- T24 a human bladder cancerous cell
- the cells were cultured in RPMI 1640 medium.
- rabbit polyclonal anti-uroprakin 1b IgG (abcam; # ab102961; dilution 1: 100), rabbit polyclonal anti-uroprakin 2 IgG (Proteintech; # 21149-1-AP; 1: 100), mice Monoclonal anti-E-cadherin IgG (BD Biosciences; # 610181; 1:50), rabbit polyclonal anti-Nanog IgG (Reprocell; # 09-0020; 1: 200), and mouse monoclonal anti-keratin 8/18 IgG (CST Japan; # 4546S; 1:50) and rabbit monoclonal anti-keratin 20 IgG (CST Japan; # 13063; 1: 100) antibody were used.
- Goat Alexa Fluor 488-labeled anti-rabbit IgG and anti-mouse IgG antibody (Life technologies; # A11034 and # 11029; 1: 500), and goat Alexa Fluor 594-labeled anti-rabbit IgG and anti-mouse IgG antibody (Life technologies; # A11037 and # 11032; 1: 500).
- the pMxs plasmid in which the human POU5F1, KLF4, MYCL, and GFP genes were integrated was donated by Professor Yamanaka (CiRA, Kyoto University, Kyoto, Japan).
- Plat-GP cells were seeded on 100 mm plates at a density of 3x10 6 cells / dish and cultured for 24 hours.
- the above 5.0 ⁇ g pMxs and 2.5 ⁇ g pCMV-VSV-G were introduced into cells using XtremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich). After 24 hours, the medium was replaced with a standard medium containing no antibiotics. The supernatant of the culture solution was cultured and used as a suspension of retrovirus.
- the medium was replaced with a retrovirus suspension filtered through a 0.45 ⁇ m pore filter (Toyo Filter Paper, ADVANTEC) and 4 ⁇ g / mL polybrene was added.
- the culture supernatant was replaced with virus-free standard medium, and 3 days later, it was replaced with CnT-Prime or "UCM" medium.
- the medium was changed every 3 or 4 days.
- the medium was replaced with "Uromedium" to differentiate DE cells into urothelial cells.
- the medium was changed once every 2-3 days and cultured for 3 days in a medium containing 10 mM rosiglitazone (Sigma-Aldrich) and not containing EGF.
- Total RNA of human urothelial cells (HUC) was purchased from ScienCell Research Laboratories (# 4325, Lot 4740).
- Total RNA of human small intestine and liver was purchased from Clontech Laboratories (# 636539, Lot 1611206A; # 636551, Lot 1703002).
- Total RNA of human salivary glands was purchased from BioChain (R1234212-10, Lot B111155).
- cDNA was synthesized using ReverTra Ace qPCR (Toyobo Life Science) and real-time RT-PCR was performed using StepOnePlus Real-Time PCR system (Applied Biosystems).
- the reaction mixture contains a Taqman probe (Applied Biosystems) and a Taqman Fast Advanced Master Mix (Applied Biosystems).
- % of signal-positive cells (signal-positive and Hoechst-positive cells / total Hoechst 33342-positive cells) x 100
- Total RNA of human urothelial cells (HUCs) was purchased from ScienCell Research Laboratories. The quality of each RNA sample was checked by agarose gel electrophoresis (gel concentration: 1%, voltage: 180V: migration time: 16 minutes) and Agilent 2100 analysis. The library was created and sequenced by Novogen Inc. Briefly, ribosomal RNA was removed using the Ribo-Zero kit. The mRNA was randomly fragmented by adding a fragmentation buffer, and cDNA was synthesized.
- the quality checked libraries were sequenced on the Illumina Novaseq 6000 (Illumina; paired-end, 150 bp).
- the filtered sequence data was mapped onto the Reference genome (GRCh38) using HISAT2 version 2.1.0 (https://ccb.jhu.edu/software/hisat2/index.shtml).
- the mapped sequences are feature counts (http://bioinf.wehi.edu.). Count using au / featureCounts /) (Liao, Y., Smyth, GK & Shi, W.
- featureCounts an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930 (2014)) Linked with the annotation GTF file of GENCODE (GENCODE 29; https://www.gencodegenes.org/). Analysis is iDEP8.1 (Ge, SX, Son, EW & Yao, R. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics 19, 10.1186 / s12859-018-2486-6 (2018)) was used. Read count data was normalized by edgeR by the CPM (counts per million) function, and genes not at least 0.5 counts / million per sample were excluded.
- the converted data is used for exploratory analysis (hierarchical clustering analysis and gene expression fluctuation analysis), and the unconverted data is used for GSEA (Gene Set Enrichment Analysis) pathway analysis as the fold-change value returned by DESeq2.
- GSEA Gene Set Enrichment Analysis
- Gene expression data is a KEGG pathway diagram (Kanehisa, Kanehisa, 2013) using Pathview (Luo, W. & Brouwer, C. Pathview: an R / Bioconductor package for pathway-based data integration and visualization. Bioinformatics 29, 1830-1831 (2013)).
- HTS Transwell clear 12-well plate with a 12 mm diameter pore size 0.4 ⁇ m polycarbonate membrane in the inner chamber was purchased from Corning and Easy iMatrix-511 silk (Nippi; 0.25 ⁇ g / cm 2 , 1 hour 37 ° C.) Coated with (pre-in vitro).
- P4 dUCs or aHDFs were suspended in UCM medium and seeded in an inner chamber at a density of 1x10 5 cells / well.
- HUCs were resuspended in Urolife TM D Cpmplete Medium (Urolife) and seeded in an inner chamber at a density of 1x10 5 cells / well.
- HUCs were cultured in Urolife supplemented with 1 ⁇ M rosiglitazone (PPAR ⁇ activator; Sigma-Aldrich) and 1 ⁇ M PD153035 (EGFR inhibitor; TCI) for 4 days.
- Cells were cultured for 5 days and washed with PBS (-).
- FITC-labeled dextran (Iwai Kagaku) with a molecular weight of about 4 kDa was added to the inside of the inner chamber at a concentration of 2 mg / mL.
- % (Fluorescence intensity of each well / Fluorescence intensity without FITC-labeled dextran) / (Fluorescence intensity of control well / Fluorescence intensity without FITC-labeled dextran) ⁇ 100
- Anti-asialo GM1 antibody was intraperitoneally administered to 6-8 week old anesthetized mice at a dose of 100 mg / mouse.
- a 24-gauge catheter was intubated transurethrally into the bladder and 50 ⁇ L of 1.5% H 2 O 2 solution was injected into the bladder.
- the bladder was washed with PBS (-), a solution containing 50 ⁇ L of dispase II (Fuji Film Wako Pure Chemical Industries, Ltd.) of 12,000 PU was injected into the bladder, and Easy iMatrix 511 silk was administered for 1 hour.
- aHDFs 3x10 6 cells / 100 ⁇ L PBS
- PBS (-) were injected intravesically. The urethra was sutured and removed 3 hours later to allow spontaneous urination.
- Example 1 Adult human skin fibroblasts (aHDFs) were induced to express a urothelial cell-like phenotype by introducing certain factors.
- FOXA1 (F) and IRF1 (I) are important in the development of urothelial cells.
- TP63 (T) is a transcription factor expressed in epithelial stem cells and associated with the development and differentiation of urothelial cells.
- SHH sonic hedgehog
- UPK1b is expressed not only in urothelial cells but also in corneal epithelium and conjunctival epithelium, but UPK2 has been reported to be more specific to urothelial cells (Habuka, M. et al., PLoS. One. 10, e0145301 (2015)).
- F, T, L, and K were necessary and sufficient to induce the expression of UPK1b and UPK2.
- the mRNA expression level of UPK1b was increased in the cells converted with F, L, and K, while the mRNA expression level of UPK2 was very high in the combination of T, L, and K.
- the combination of L and K and the combination of F, L and K also significantly increased the mRNA expression level of UPK2.
- Example 2 Characteristics of directly converted urothelial cells (dUCs).
- the cells converted by the method of Example 1 are represented as directly converted urothelial cells (dUC).
- dUC directly converted urothelial cells
- the detailed characteristics of the cells were investigated.
- cells converted with F, T, L, and K markedly expressed UPK1b 8 days after gene transfer, and expression peaked after 15 days (Fig. 8a).
- UPK2 mRNA expression began to rise after 11 days in FTLK-converted cells and peaked after 21 days. The formation of epithelial cell colonies was observed after 11 days and then increased (Fig. 8b).
- dUCs prepared by direct conversion and urothelial cells (iUCs) derived from iPS cells induced by the method of Osborne et al. With some modifications were compared (Fig. 9A).
- the dUCs strongly expressed the UPK1b and UPK2 genes, while the iUCs strongly expressed the UPK1a and UPK3a genes (Fig. 9B). This strongly suggested that dUCs resemble more immature urothelial cells than iUCs.
- Example 3 Pluripotency, liver and small intestine markers were not expressed in dUCs. Many in F, T, L, K-introduced cells during the conversion process to rule out the possibility that fibroblasts were temporarily converted to iPS cells or endoderm progenitor cells, resulting in urothelial cells. The expression of potency markers and the expression of non-urothelial endoderm markers were examined. As a result, in F, T, L, and K-introduced cells, gene expression of NANOG, POU5F1, LIN28, and SOX2 was not observed during the culture period. Similarly, the NANOG protein was not observed (Fig. 10).
- F, T, L, and K-introduced cells did not express mRNA for the small intestinal epithelial cell marker CDX2 and the hepatocyte marker albumin (ALB) at any observation time (FIG. 11).
- F, T, L, and K-introduced cells did not express mRNA of CD31, which is a mesenchymal-derived endothelial cell marker, and AQP5, which is a marker of ectoderm-derived salivary gland (Fig. 11). Therefore, it was confirmed that the F, T, L, and K-introduced cells did not undergo the pluripotency stage in the process of conversion to dUCs and did not convert to cells derived from other endoderm. It was also confirmed that the cells were not converted to mesenchymal or ectoderm cells.
- Example 4 dUCs can be selectively grown by subculture.
- the dUC colonies formed by 21 days after gene transfer did not grow actively even when continuously cultured on the same medium.
- cells were started to be cultured in the first medium (generation number 0, P0) and then subcultured to a new medium (generation number 1, P1).
- generation number 0, P0 generation number 1
- DUCs of P0 prepared in CnT-Prime medium were newly cultured in the same medium, almost no epithelial cell colonies were formed, and only a few non-epithelial cells proliferated.
- "Uromedium” did not form paving stone-like colonies in the next generation (Fig. 12).
- the concentration of the medium component contained in "Uromedium” was improved and the composition was determined. As shown in FIG. 13A, the concentrations of insulin and hydrocortisone were not definitive.
- 1-10 mM IBM X played a beneficial role, including the expression of UPK2 mRNA in F, L, T, and K-introduced cells. In the hydrocortisone-free medium and the medium containing 10 mM IBM X, F, L, T, and K-introduced cells formed epithelial cell colonies (Fig. 13B).
- urothelial cell conversion and maintenance medium has the following composition.
- RNA sequencing further confirmed the characteristics of P2 dUCs as urothelial cells.
- the similarity between P2 dUCs and urothelial cells was analyzed by RNA sequencing.
- Gene expression profiling analysis by hierarchical clustering of the entire genome revealed that dUCs are more similar to HUCs than aHDF (Fig. 16A).
- FIG. 16B shows data for the 50 most variable genes. The results show that genes related to urothelial cells, including keratin (KRT8, 18, 5, 17, 6A, 14), which is rarely expressed in fibroblasts, and RAB27B, which is involved in the transport of uroprakin to the upper cell, Both dUCs and HUCs have been shown to be characteristically expressed.
- fibroblast-related genes FAP, COL16A1, COL1A2, DPT, PTX3, etc.
- FAP fibroblast-related genes
- COL16A1, COL1A2, DPT, PTX3, etc. genes associated with urothelial cells rather than fibroblasts
- HUCs and dUCs showed similar expression patterns (Fig. 17A).
- dUCs expressed KLF5 one of the important factors involved in the development of bladder epithelial cells
- KEGG pathway analysis also revealed that dUCs strongly expressed genes involved in the formation of interepithelial tight junctions between cells (Fig. 17B). These genes are also rarely expressed in fibroblasts.
- Example 7 dUCs may contribute to the regeneration of damaged urothelial cells in vivo.
- aHDFs introduced with F, T, L, and K converted to dUCs in vivo and contributed to the regeneration of urothelial cells.
- the aHDFs into which the F, T, L, K and GFP genes were introduced were converted to GFP-labeled dUCs after culturing in CnT-Prime medium for 21 days (Fig. 18a, b).
- aHDFs were transplanted into the bladder of mice damaged by interstitial cystitis 4 days after F, T, L, K, G gene transfer.
- GFP-labeled cells were present in the mucosal epithelium of the bladder and expressed UPK1b, UPK2, E-cadherin, and keratin 8/18 (Fig. 18d).
- KRT20 which is said to be expressed in finally differentiated umbrella cells, was also expressed.
- transplantation of GFP-labeled aHDFs into the bladder did not result in GFP-positive and E-cadherin-positive cells (Fig. 18d). Therefore, aHDF introduced with F, L, T, K, and G was converted to dUCs in vivo, localized in the injured bladder epithelium, and involved in tissue regeneration.
- Example 8 Examination of the presence or absence of tumorigenicity of dUCs The potential tumorigenicity of dUCs was investigated. Previously tested, F, L, T, and K-introduced aHDFs did not form tumors after subcutaneous inoculation of immunodeficient mice (Fig. 19). On the other hand, T24 bladder cancer cells formed tumors.
- Example 9 It was confirmed that the urothelial cell-like phenotype was expressed by introducing a specific factor into aHDFs in the same manner as in Example 1. The results are shown in FIG. It was shown that F, T, L, K or a combination thereof is effective in inducing the expression of UPK1b and UPK2.
- urothelial cells can be prepared from somatic cells in a short period of time by direct reprogramming. Since these urothelial cells can be easily derived from the somatic cells of the person to be transplanted, problems such as immunological rejection do not occur even when the obtained urothelial cells are transplanted. In addition, since urothelial cells can be directly induced from somatic cells without passing through iPS cells or ES cells, problems caused by pluripotent stem cells such as canceration can be avoided.
- This application is based on Japanese Patent Application No. 2019-159070 filed in Japan (Filing date: August 30, 2019), the contents of which are incorporated herein by reference in its entirety.
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Abstract
Description
また、間質性膀胱炎の一つであるハンナ型間質性膀胱炎の現在の治療として、膀胱内にDMSOやキシロカインを注入する方法、膀胱を無理やり拡張させる方法(膀胱拡張術)が実施されている。しかしながら、短期的な成績はあるものの長期的な成績は乏しく、根本的治療法がないのが現状である。
即ち、本発明は以下の通りである。
[1]哺乳動物の体細胞に、外来性因子として、
FOXA1(Forkhead box A1)遺伝子若しくはその発現産物、
TP63(tumor protein P63)遺伝子若しくはその発現産物、
MYCL(L-Myc)遺伝子若しくはその発現産物、及び
KLF4(Kruppel-like factor 4)遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を導入する工程を含む、尿路上皮細胞を誘導する方法。
[2]外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、上記[1]記載の方法。
[3]外来性因子が、以下より選択される、上記[1]記載の方法;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
[4]前記体細胞がヒト線維芽細胞である、上記[1]~[3]のいずれかに記載の方法。
[5]哺乳動物の体細胞に由来し、外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を有する、尿路上皮細胞。
[6]外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、上記[5]記載の細胞。
[7]外来性因子が、以下より選択される、上記[5]記載の細胞;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
[8]前記体細胞がヒト線維芽細胞である、上記[5]~[7]のいずれかに記載の細胞。
[9]外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を含む、哺乳動物の体細胞を尿路上皮細胞にコンバートする為の剤。
[10]外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、上記[9]記載の剤。
[11]外来性因子が、以下より選択される、上記[9]記載の剤;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
[12]前記体細胞がヒト線維芽細胞である、上記[9]~[11]のいずれかに記載の剤。
[13]外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を含む、哺乳動物の体細胞を尿路上皮細胞にコンバートする為のベクター。
[14]外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、上記[13]記載のベクター。
[15]外来性因子が、以下より選択される、上記[13]記載のベクター;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
[16]前記体細胞がヒト線維芽細胞である、上記[13]~[15]のいずれかに記載のベクター。
[17]上記[1]~[4]のいずれかに記載の方法で得られる細胞、上記[5]~[8]のいずれかに記載の尿路上皮細胞、上記[9]~[12]のいずれかに記載の剤、又は上記[13]~[16]のいずれかに記載のベクターを含む、泌尿器系の疾患の治療剤。
[18]泌尿器系の疾患が、尿路疾患である、上記[17]に記載の治療剤。
[19]尿路疾患が、膀胱がん、過活動膀胱、膀胱の先天異常、膀胱損傷、間質性膀胱炎、神経因性膀胱及び萎縮膀胱からなる群より選択される少なくとも1種である、上記[18]に記載の治療剤。
[20]3-イソブチル 1-メチルキサンチン(IBMX)及び上皮成長因子(EGF)を含むことを特徴とする、尿路上皮細胞誘導用培地。
尿路上皮細胞(移行上皮細胞ともいう)は、腎杯、腎盂から尿管及び膀胱を経て尿道に至るまでの尿路の内側を覆っている細胞であり、一実施形態では、尿路上皮細胞は、膀胱に由来する上皮細胞であってもよい。
本発明において、ダイレクト・リプログラミングの対象となる体細胞としては、特に限定されるものではなく、哺乳動物由来であればよい。尿路上皮細胞を生体に移植する場合には、移植される被験体由来の体細胞(自家細胞)を用いることが、感染や拒絶応答等の危険を低減させるために好ましい。しかしながら、目的によっては、自家細胞でなく、他人や他の動物の体細胞からあらかじめ準備しておいた尿路上皮細胞を移植に用いることができる。またはあらかじめ準備しておいた他人や他の動物の体細胞から尿路上皮細胞を作り、移植に用いることができる。すなわち、尿路上皮細胞のバンクを作っておき移植目的に供することができる。このような場合、拒絶応答等の危険を低減させるために、あらかじめMHCをタイピングしておくことができる。また、あらかじめ尿路上皮細胞の細胞特性や造腫瘍性などを確認しておくことができる。
本発明の方法では、体細胞に、外来性因子として、FOXA1(Forkhead box A1)遺伝子若しくはその発現産物、TP63(tumor protein P63)遺伝子若しくはその発現産物、MYCL(L-Myc)遺伝子若しくはその発現産物、及びKLF4(Kruppel-like factor 4)遺伝子若しくはその発現産物からなる少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てを導入する。ここで、「発現産物」としては、FOXA1遺伝子、TP63遺伝子、MYCL遺伝子及びKLF4遺伝子のmRNA又はタンパク質が挙げられる。
本発明の方法において、体細胞に導入される外来性因子の好ましい組み合わせの一実施態様は、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物の組み合わせを含む。
外来性因子が以下より選択される本発明の方法もまた、好ましい一実施態様である。
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
いずれもUPK2mRNAの発現を増強させることができる。
ヒトFOXA1遺伝子cDNA配列:BC_033890、NM_004496(例えば、BC_033890.1、NM_004496.5)、
ヒトFOXA1タンパク質アミノ酸配列:NP_004487(例えば、NP_004487.2)、
ヒトTP63遺伝子cDNA配列:BC_039815、NM_003722(例えば、BC_039815.1、NM003722.5)、
ヒトTP63タンパク質アミノ酸配列:NP_003713(例えば、NP_003713.3)、
ヒトMYCL遺伝子cDNA配列:NM_001033081、NM_001033082、NM_005376(例えば、NM_001033081.2、NM_001033082.2、NM_005376.4)、
ヒトMYCLタンパク質アミノ酸配列:NP_001028253.1、NP_001028254.2、NP_005367.2(例えば、NP_001028253、NP_001028254、NP_005367)、
ヒトKLF4遺伝子cDNA配列:BC_029923、NM_001314052(例えば、BC_029923.1、NM_001314052.2)、
ヒトKLF4タンパク質アミノ酸配列:NP_001300981(例えば、NP_001300981.1)。
本発明の方法は、特定の遺伝子を選択する以外は、公知のダイレクト・リプログラミングの手法に準じて行うことができ、例えば以下のいずれかの文献の方法に準じて行うことができる:
1: Direct Reprogramming of Fibroblasts into Functional Cardiomyocytesby Defined Factors; Masaki Ieda, Ji-Dong Fu, Paul Delgado-Olguin, Vasanth Vedantham, Yohei Hayashi, Benoit G. Bruneau, and Deepak Srivastava Cell 142: 375-386, 2010.
2: Direct conversion of fibroblasts to functional neurons by defined factors. Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Kokubu, Thomas C. Sudhof& Marius Wernig. Nature 46.
3: 1035-1041, 20103: Induction of human neuronal cells by defined transcription factors. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Sudhof TC, Wernig M. Nature 476: 220-223, 2011.
4: Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors Kunihiko Hiramatsu, Satoru Sasagawa, Hidetatsu Outani, Kanako Nakagawa, Hideki Yoshikawa, and Noriyuki Tsumaki, Journal of Clinical Investigation, 121: 640-657, 2011.
5: Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Pengyu Huang, Zhiying He, Shuyi Ji, Huawang Sun, Dao Xiang, Changcheng Liu, Yiping Hu, XinWang & Lijian Hui,. Nature 475:386-389, 2011.
6: Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Sayaka Sekiya & Atsushi Suzuki. Nature 475:390-393, 2011.
7: Direct conversion of human fibroblasts into functional osteoblasts by defined factors. Yamamoto K, Kishida T, Sato Y, Nishioka K, Ejima A, Fujiwara H, Kubo T, Yamamoto T, Kanamura N & Mazda O. Proc Natl Acad Sci USA. 112:6152-6157, 2015.
8: Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes. Kishida T, Ejima A, Yamamoto K, Tanaka S, Yamamoto T, Mazda O. Stem Cell Reports. 5: 569-581, 2015.
9: Generation of directly converted human osteoblasts that are free of exogenous gene and xenogenic protein. Yamamoto K., Sato Y., Honjo K., Ichioka H., Oseko F., Sowa Y., Yamamoto T., Kanamura N., Kishida T., Mazda O. J Cell Biochem 117:2538-2545, 2016.
10: 国際公開公報WO2014/010746
上記の文献1~10の内容は本明細書に参考として援用される。
本発明の方法において、哺乳動物の分化した体細胞を、遺伝子の導入後、培地中で培養することができる。例えば、インビトロで尿路上皮細胞を誘導(調製)する場合の好適な態様である。
本発明の方法で用いる培地は、特に限定されない。MEM(Modified Minimum Essential Medium )、DMEM(Dulbecco's Modified Eagle's Medium)、EMEM(Eagle's minimal essential medium)、αMEM(alpha Modified Minimum Essential Medium)などの通常の液体培地を用いることができる。必要に応じて、血清成分(Fetal Bovine Serum(FBS)、Human Serum(HS))、ストレプトマイシン、ペニシリンなどの抗菌薬、非必須アミノ酸(Non-Essential Amino Acids;NEAA)等の成分を添加することができる。
好ましくは、本発明の尿路上皮細胞誘導培地は、3-イソブチル 1-メチルキサンチン(IBMX)及び上皮成長因子(EGF)を含む。培地中のIBMXの濃度は通常、1μM~10mMであるが、尿路上皮細胞の選択的な維持増殖の為には1mM~10mMのIBMXが培地中に含まれていることが好ましい。培地中のEGFの濃度は通常、0.001~1ng/mL、好ましくは0.01~1ng/mLである。これらの成分を含有することによって実施例に示されるように、線維芽細胞から尿路上皮細胞へのコンバート及びそれに続く尿路上皮細胞の選択的な維持増殖がより効率的になる。
本発明の尿路上皮細胞誘導培地の好ましい一例として、下記組成の培地が挙げられる(本明細書中、「UCM培地」とも称する)。
60μMカルシウム、
60μg/mLウシ脳下垂体抽出液、
5μg/mLヒト遺伝子組み換えインスリン、
ゲンタマイシン・アンフォテリシン、
2%FBS、
0.01ng/mLのヒト遺伝子組み換えEGF、
1mMのIBMX、および
1μMのトラニルシプロミン
を添加したEpiLifeTM培地。
(EpiLifeTM培地:ケラチノサイト培養用の培地)
かくして、体細胞から尿路上皮細胞が誘導される。尿路上皮細胞が得られたことは、例えばウロプラキン(Uroplakin1b;UPK1)、Uroplakin2(UPK2)、CK5,CK17の遺伝子発現、Asymmetric Unit Membraneの形成等によって検出することができる。
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
ここで、「外来性」とは、主に上記の導入手段の結果導入された遺伝子又はその発現産物の態様であって、天然の態様とは異なる態様を指す。例えば、天然のプロモーター以外のプロモーターに発現を制御される遺伝子、天然以外の染色体上の位置、若しくは、染色体外に存在する遺伝子の態様などが挙げられる。
治療する対象となる具体的な疾患としては、膀胱がん、過活動膀胱、膀胱の先天異常(例、膀胱外反症、膀胱憩室、尿膜管の異常)、膀胱損傷、間質性膀胱炎(ハンナ型及び非ハンナ型)、神経因性膀胱、萎縮膀胱等が挙げられる。
前述するように、外来性因子として、FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物からなる群より選択される少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てを体細胞に導入することにより、尿路上皮細胞を誘導できる。従って、本発明は、さらに、FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物からなる群より選択される少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てを含む、尿路上皮細胞を誘導するための組成物を提供する。当該尿路上皮細胞を誘導するための組成物は、体細胞から尿路上皮細胞を誘導するために使用される因子を含むものであり、FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物からなる群より選択される少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てが体細胞に導入可能な形態で含まれていることが望ましい。導入される遺伝子の種類や数によっても異なるが、別の一実施態様においては、外来性因子は、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む。さらに別の一実施態様においては、以下の遺伝子若しくはその発現産物、又はそれらの組み合わせを含む。
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。
上記遺伝子が体細胞に導入可能な形態として、具体的には、上記遺伝子が組み込まれたベクターが例示される。ここで、上記遺伝子は、各々別のベクターに組み込まれていてもよく、1つのベクターに2種以上の遺伝子が同時に組み込まれていてもよい。
尿路上皮細胞の欠損部位に存在する体細胞(例、線維芽細胞)に、FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物からなる群より選択される少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てを導入することで、当該損傷部位において尿路上皮細胞をダイレクト・リプログラミングによって誘導し、もって尿路上皮細胞の治療や尿路上細胞の再生に寄与することができる。FOXA1遺伝子若しくはその発
現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物の導入においては、上記の本発明の組成物を好適に使用することができる。
1: Ieda M. Heart regeneration using reprogramming technology. Proc Jpn Acad Ser B Phys Biol Sci. 2013;89(3):118-28. Review.
2: Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010 Aug 6;142(3):375-86.
3: Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 2012 May 31;485(7400):593-8.
また、膀胱内や腎盂内等の管腔内にFOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物からなる群より選択される少なくとも1種、好ましくは2種、より好ましくは3種、一層好ましくは4種全てを注入することにより行うことができる。尿道カテーテル等で膀胱内や腎盂内に上記遺伝子若しくはその発現産物あるいはそれらの組み合わせを注入し、膀胱内や腎盂内に存在する体細胞と該遺伝子若しくはその発現産物あるいはそれらの組み合わせとを接触させ体細胞を尿路上皮細胞へと誘導する。
<<略語一覧>>
aHDF(adult Human Dermal Fibroblast;成人ヒト皮膚線維芽細胞)
UC(urothelial cell)
HUC(Human urothelial cell)
HUVEC(Human Umbilical Vein Endothelial Cell)
dUC(ダイレクト・コンバージョンされたUC)
FOXA1(Forkhead box A1;本明細書中「F」とも称する)
TP63(tumor protein P63;本明細書中「T」とも称する)
MYCL(L-Myc;本明細書中、「L」とも称する)
KLF4(Kruppel-like factor 4;本明細書中、「K」とも称する)
IRF1(Interferon regulatory factor 1;本明細書中、「I」とも称する)
GFP(green fluorescent protein)
1.細胞培養
aHDFsをラミニンコーティングした12-wellプレート、あるいはラミニンコーティングしていない12-wellのプレートに播種し、レトロウイルスベクターで遺伝子導入を行った。細胞をCnT-Prime培地または他の培地で培養し、21日後に位相差顕微鏡で観察、リアルタイムPCR、免疫細胞化学の解析を行った。細胞をフレッシュな培養プレートで継代し、RNAシーケンスと浸透アッセイに用いた。In vivoの実験では、F、T、L、Kを導入された細胞を、間質性膀胱炎マウスの損傷した膀胱腔内に、カテーテルを通じて経尿道的に移植した。
「標準培地」として、MEM非必須アミノ酸、100mMピルビン酸ナトリウム、100U/mLペニシリン-ストレプトマイシンを添加した10%FBS(Gibco)添加高グルコースDMEMを用いた。
「CnT-Prime(登録商標)」培地は、CeLLnTECから購入した。
「Uromedium」培地は、オズボーンらの方法(Osborn SL, (2014) Stem Cell Transl Med 3(5):610-619.)に従い作成した。要すれば「Uromedium」培地は、60μMのカルシウム(Gibco;MEP1500CA)と60μg/mLのウシ脳下垂体抽出液(Gibco)、5μg/mLのヒト遺伝子組み換えインスリン(Diagnocine)、500ng/mLのヒドロコルチゾン(東京化成工業)、ゲンタマイシン・アンフォテリシン(Gibco)、2%FBS、0.1ng/mLヒト遺伝子組み換え上皮成長因子(EGF)(RSD)、100μM 3-イソブチル 1-メチルキサンチン(IBMX)(Sigma-Aldrich)を添加したEpiLifeTM培地からなる。
「UCM」培地は、60μMカルシウムと60μg/mLウシ脳下垂体抽出液、5μg/mLのヒト遺伝子組み換えインスリン、ゲンタマイシン・アンフォテリシン、2%FBS、0.01ng/mLのヒト遺伝子組み換えEGF、1mMのIBMXおよび1μMのトラニルシプロミン(Abcam)を添加したEpiLifeTM培地からなる。
aHDFsと、Plat-GP細胞は、ScienCell Research LaboratoriesとCell Biolabからそれぞれ購入し、標準培地で培養した。継代数10世代以下のaHDFsが実験で使用された。HUCsは倉敷紡績株式会社から購入した(KP-4309;Lot04101)。人工多能性幹細胞(iPS細胞)は、JCRB Cell Bankから購入し、既報(Nakagawa M,et al., (2014) Sci Rep 4(3594):10.1038/srep03594.1)に従って維持された。要すれば、iPS細胞は、Easy iMatrix-511 silk(ニッピ;0.25μg/cm2;事前に37℃で1時間処理された)でコートされたプレート上、StemFit培地(味の素;AK02N)中で培養した。継代のために、iPS細胞を解離しRock阻害剤(ナカライテスク;Y-27632)を含むStemFit培地で単一細胞の懸濁液とし、新しい培養プレートに再播種した。翌日、Rock阻害剤を含まない新しいStemFit培地に交換し、2日おきに培地交換した。継代は、iPS細胞が80-90%のコンフルエントに達した時に行った。ヒト膀胱がん化細胞であるT24は、芦原教授(京都薬科大学、京都、日本)から提供され、10%FBS、MEM非必須アミノ酸、100mMのピルビン酸ナトリウムと100U/mLのペニシリン・ストレプトマイシンを添加したRPMI1640培地で培養した。
免疫抗体染色の1次抗体として、ウサギポリクローナル抗ウロプラキン1b IgG(abcam; #ab102961; dilution 1:100)、ウサギポリクローナル抗ウロプラキン2 IgG(Proteintech; #21149-1-AP; 1:100)、マウスモノクローナル抗E-カドヘリン IgG(BD Biosciences; #610181; 1:50)、ウサギポリクローナル抗Nanog IgG(Reprocell; #09-0020; 1:200)、およびマウスモノクローナル抗ケラチン8/18 IgG(CST Japan; #4546S; 1:50)、及びウサギモノクローナル抗ケラチン20 IgG(CST Japan; #13063; 1:100)抗体を使用した。2次抗体は以下の抗体を用いた。ヤギAlexa Fluor 488標識抗ウサギIgGと抗マウスIgG抗体(Life technologies; #A11034 and #11029; 1:500)、およびヤギAlexa Fluor 594標識抗ウサギIgGと抗マウスIgG抗体(Life technologies; #A11037 and #11032; 1:500)。
FOXA1、IRF1、TP63、ソニックヘッジホッグ遺伝子の全長cDNAクローンはDNAFORM Libraryから購入し、KOD-Plus-Neo DNAポリメラーゼ(東洋紡)を用いてRT-PCRによりコーディング領域を増幅した。プライマー配列は以下の通り。
FOXA1
5’-CAGCAGTGTGGTGGTACGGGATGTTAGGAACTGTGAAGATG-3’(配列番号1)
5’-ACCGGCGCTCAGCTGGCTAGGAAGTGTTTAGGACGGG-3’(配列番号2)
IRF1
5’-CAGTGTGGTGGTACGGGATGCCCATCACTCGGATGCGC-3’(配列番号3)
5’-ACCGGCGCTCAGCTGGACCGGCGCTCAGCTGG-3’(配列番号4)
TP63
5’-CAGTGTGGTGGTACGGGATGAATTTTGAAACTTCACGG-3’(配列番号5)
5’-ACCGGCGCTCAGCTGGCTATCACTCCCCCTCCTCTTT-3’(配列番号6)
ソニックヘッジホッグ
5’-CAGTGTGGTGGTACGGGATGCTGCTGCTGGCGAGATGT-3’(配列番号7)
5’-ACCGGC GCTCAGCTGGCTAGCTGGACTTGACCGCCAT-3’(配列番号8)
増幅したDNAフラグメントは、Gene Art System(Invitrogen)を用いてPMxsレトロウイルスベクター(Cell Biolabs)でクローニングされた。ヒトPOU5F1、KLF4、MYCL、GFP遺伝子が組み込まれているpMxsプラスミドは、山中教授(CiRA、京都大学、京都、日本)から供与された。Plat-GP細胞を3x106cells/dishの密度で100mmのプレートに播種し、24時間培養した。上述の5.0μgのpMxsと2.5μgのpCMV-VSV-Gを、XtremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich)を用いて細胞に導入した。24時間後、抗生物質を含まない標準培地に交換した。培養液の上清みを培養し、レトロウイルスの懸濁液として用いた。
aHDFsを、アテロコラーゲン(KOKEN;I-PC30)あるいはポリ-L-リジン(ScienCell)あるいはラミニン(Easy iMatrix-511 silk)でコーティングした12-wellプレートに、3x104cells/wellの密度で播種した。24時間後、0.45μm孔フィルター(東洋濾紙、ADVANTEC)でフィルタリングしたレトロウイルス懸濁液と培地を交換し、4μg/mLのポリブレンを添加した。翌日、培養上清液をウイルスを含まない標準培地と交換し、3日後、CnT-Primeあるいは「UCM」培地と交換した。培地は、3日あるいは4日おきに交換した。
dUCsが60-80%のコンフルエントになるか、前回の継代から14日経過した時にdUCsを継代した。トリプシン/EDTA(ナカライテスク)とCell Scraper (イワキ)を用いてdUCsを剥離し、1x104cells/wellの密度でラミニンでコーティングされた12-wellプレートに播種し、「UCM」培地で培養した。
iPS細胞を既報(Osborn SL, et al., (2014) Stem Cell Transl Med 3(5):610-619.)の方法に少しの改変を加え尿路上皮細胞にコンバートした。要すれば、ラミニンでコーティングされた6-wellプレートに、2x104cells/wellの密度でiPS細胞を播種し、60-70%コンフルエントになるまで培養し、培地を、2%FBS、GlutaMAX (Gibco)、100U/mLペニシリン・ストレプトマイシン、5μMIDE1 (Cayman)を添加したRPMI1640に交換した。培地は、1-2日おきに新しいものと交換した。内胚葉細胞(DE)へ分化誘導した9日後に、培地を「Uromedium」に交換し、DE細胞を尿路上皮細胞に分化させた。18日間の培養期間中、培地は2-3日に1回交換し、10mMロジグリタゾン(Sigma-Aldrich)を含みEGFを含まない培地で3日間培養した。
RNeasy Mini Kit (Qigen)を用いて、製造元のプロトコルに従い全RNAを抽出した。ヒト尿路上皮細胞(HUC)の全RNAはScienCell Research Laboratoriesから購入した(#4325、Lot 4740)。ヒト小腸、肝臓の全RNAはClontech Laboratoriesから購入した(#636539, Lot 1611206A;#636531, Lot 1703002)。ヒト唾液腺の全RNAはBioChain(R1234212-10, Lot B111155)から購入した。内皮細胞の全RNAはヒト臍帯静脈内皮細胞(HUVEC; PromoCell; C-12200)から抽出した。cDNAを、ReverTra Ace qPCR (東洋紡ライフサイエンス)を用いて合成し、StepOnePlus Real-Time PCRシステム(Applied Biosystems)を用いてリアルタイムRT-PCRを行った。反応混合液は、Taqmanプローブ(Applied Biosystems)とTaqman Fast Advanced Master Mix (Applied Biosystems)を含む。相対的なmRNA発現レベルは下式により求めた。
相対的mRNAレベル(fold)=(サンプル中のターゲット遺伝子mRNA/サンプル中のβ-actin遺伝子mRNAレベル)/(コントロールサンプル中のターゲット遺伝子mRNAレベル/コントロールサンプル中のβ-actin遺伝子mRNAレベル)
12-wellプレートで培養した細胞をPBS(-)で洗浄し、4%パラホルムアルデヒド(PFA)で30分間固定後、0.2%Triton-X 100(ナカライテスク)で15分間、室温で膜透過処理を行った。Blocking One Histo (ナカライテスク)を室温30分間添加しバックグラウンドのブロッキングを行い、1次抗体と4℃で一晩反応させた。洗浄後、2次抗体を細胞に加え、遮光し1時間室温で反応させた。細胞を洗浄し、核をヘキスト33342(DOJINDO)で5分染色した。観察はBZ-X (Keyence)で行った。各シグナルの陽性、陰性細胞をBZ-X Analyzer software (Keyence)でカウントした。シグナル陽性細胞の割合は下式により算出した。
% of シグナル陽性細胞=(シグナル陽性かつヘキスト陽性細胞/全ヘキスト33342陽性細胞)x 100
aHDFsとP2 dUCs(UCMで培養)から、RNeasy Mini Kit (QIAGEN)を用いて、製造元のプロトコルに従い全RNAを抽出した。ヒト尿路上皮細胞(HUCs)の全RNAはScienCell Research Laboratoriesから購入した。各RNAサンプルの質は、アガロースゲル電気泳動(ゲル濃度:1%、電圧:180V:泳動時間:16分)とAgilent 2100の解析でチェックした。ライブラリーの作成とシーケンスはNovogen Inc.で行った。簡単には、Ribo-Zero kitを用いてリボソームRNAを除去した。mRNAをフラグメンテーションバッファーを加えランダムに断片化し、cDNAの合成を行った。品質チェックされたライブラリーをIllumina Novaseq 6000 (Illumina; paired-end, 150 bp)でシーケンスした。フィルターされた配列データは、HISAT2 version 2.1.0 (https://ccb.jhu.edu/software/hisat2/index.shtml) を用いてReference genome(GRCh38)上にマッピングした。Bedtools (https://bedtools.readthedocs.io/en/latest/)で同定された配列結果から、残存リボソームRNAを除去した後に、マッピングされた配列をfeaturecounts (http://bioinf.wehi.edu.au/featureCounts/)(Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930 (2014))を用いてカウントし、GENCODE (GENCODE 29; https://www.gencodegenes.org/)のアノテーションGTFファイルと紐づけた。解析は、iDEP8.1(Ge, S. X., Son, E. W. & Yao, R. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics 19, 10.1186/s12859-018-2486-6 (2018))を用いて行った。リードカウントデータは、CPM(counts per million)ファンクションによりedgeRで正規化され、少なくとも1サンプル当たり0.5カウント以上/millionでない遺伝子は排除した。変換されたデータは探索解析(階層クラスタリング解析と遺伝子発現変動解析)に用いられ、変換しなかったデータは、DESeq2で返されたfold-change値としてGSEA (Gene Set Enrichment Analysis)のパスウェイ解析に用いられた。遺伝子発現データはPathview(Luo, W. & Brouwer, C. Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics 29, 1830-1831 (2013))を用いたKEGGパスウェイダイアグラム(Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45, D353-D361 (2017))上で可視化された。
内側のチャンバーに直径12mmの孔径0.4μmのポリカーボネート膜を有するHTS Transwell clear 12-wellプレートをコーニングから購入し、Easy iMatrix-511 silk (ニッピ;0.25μg/cm2、1時間37℃でプレインキュベート)でコーティングした。P4 dUCsあるいはaHDFsをUCM培地に懸濁し、inner chamberに1x105cells/wellの密度で播種した。HUCsはUrolifeTMD Cpmplete Medium(Urolife)に再懸濁し、inner chamberに1x105cells/wellの密度で播種した。最終分化させる為に、一部のHUCsを1μMのロジグリタゾン(PPARγ活性化剤;Sigma-Aldrich)及び1μMのPD153035(EGFR阻害剤;TCI)を添加したUrolife中で4日間培養した。細胞を5日間培養し、PBS(-)で洗浄した。分子量約4kDaのFITC標識デキストラン(岩井化学)をinner chamberの内部に2mg/mLの濃度で加えた。20分間遮光下で培養し、outer chamber内の上清液を96-wellプレートに移し、各wellの蛍光強度をSectraMax M2(Molecular Devices)を用いて、蛍光533nm、励起485nmで蛍光強度を測定した。outer chamberに漏れ出したFITC標識デキストランの割合の、細胞を播種しなかったコントロールを100%とした相対値(%)を、下式により算出した。
%=(各wellの蛍光強度/FITC標識デキストラン非添加の蛍光強度)/(コントロールwellの蛍光強度/FITC標識デキストラン非添加の蛍光強度)×100
全ての動物実験は、大学の動物実験委員会(M30-281)で承認された。動物の取り扱いは大学のガイドラインに従った。メスのNOG/BALB-Rag2nullIL-2Rγnull/NSG (NOG/SCID) (NOG/SCID)マウスは日本クレアより購入した。間質性膀胱炎マウスモデルと膀胱内細胞移植方法は既報(Homan T, et al., (2013) J Pharmacol Sci 121(4):327-337.、Shimizu T, et al., (2018) Oncoimmunology 7(5):e1424671.)の方法に記載されている方法に少しの修正を加え行った。6-8週齢の麻酔下のマウスに、抗asialo GM1抗体を100mg/マウスの用量で腹腔内投与した。同時に、24ゲージカテーテルを膀胱内に経尿道的に挿管し、1.5%のH2O2液50μLを膀胱内に注入した。1時間後、膀胱をPBS(-)で洗浄し、12,000PUのdispase II(富士フィルム和光純薬株式会社)50μLを含む溶液を膀胱内に注入し、Easy iMatrix 511 silkを1時間投与した。F、T、L、Kを導入されたaHDFsを標準培地で4日間培養し、PBS(-)に3x106/100μLの濃度で懸濁後、膀胱内に移植した(それぞれn=4)。その他の群は、遺伝子導入していないaHDFs(3x106cells/100μL PBS)あるいはPBS(-)のみを膀胱内に注入した。尿道を縫合し、3時間後に抜糸し、自発的な排尿が出来るように処置した。
6-8週齢のメスNOG/SCIDマウスを麻酔し、100mgの抗asialo GM1抗体を腹腔内投与した。F、T、L、Kを導入したaHDFsを標準培地で4日間培養後、Matrigel (Corning)と混合し、1x107/100μLの濃度でマウス皮下に投与した。T24細胞と遺伝子導入していないaHDFsはそれぞれ陽性、陰性コントロールとして用いた。投与部位の組織膨張の径、または腫瘍ができた場合には腫瘍の径をノギスで1週ごとに10週間測定し、体積を計算した。
細胞移植後7日目にマウスの膀胱を摘出した。ホルムアルデヒドで固定後、幾つかの臓器は30%スクロース/PBS(-)で置換した。その他の部位はパラフィン包埋した。サンプルを5-10μmの厚さで薄切した。内因性のペルオキシダーゼ活性を3%H2O2で5分間ブロッキングし、Protein Block (Dako)で30分間インキュベートした。洗浄後、組織切片を1次抗体と4℃で一晩反応させた。組織を洗浄し、ヤギAlexa Fluor 594標識抗ウサギIgG2次抗体、あるいは抗マウスIgG2次抗体と室温で60分反応させた。3回洗浄後、組織切片をDAPI入りのVECTASHIELD mounting medium (Vector Laboratories)で封入した。蛍光観察は、BZ-X (Keyence)を用いて行った。
各データは平均±SDで表記した。統計的有意差は、Student's t-testまたはone-way ANOVAとポストホックのTukey-Kramerテストで検定した。統計的有意差はP<0.05に設定した。全ての解析はEZR(Kanda, Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone. Marrow. Transplant. 48, 452-458 (2013))で解析した。
成人ヒト皮膚線維芽細胞(aHDFs)は、特定の因子を導入することにより尿路上皮細胞様の表現型を発現するよう誘導された。
最初に、3つの転写因子に注目した。FOXA1(F)、IRF1(I)は、尿路上皮細胞の発生において重要である。一方、TP63(T)は上皮幹細胞において発現し、尿路上皮細胞の発生・分化に関連した転写因子である。また、尿路上皮細胞の基底膜細胞で発現し、再生過程における細胞増殖に重要であるシグナルを調節するソニックヘッジホッグ(SHH)遺伝子(H)を加えた。これらの遺伝子をレトロウイルスベクターを用いて、成人ヒト皮膚線維芽細胞(aHDFs)に導入し、該細胞を、細胞の維持に適切と思われる期間(21日間)、CnT-Prime培地で培養した(図1)。FIT、またはFIHを導入した細胞では、尿路上皮細胞の発生マーカーであるウロプラキン(UPK1b)が僅かに発現した(図2A)。どちらの細胞も、尿路上皮細胞形態様のコロニーは形成しなかった(図2B)。次に、初期化遺伝子である3つの遺伝子、すなわちPOU5F1 (P)、KLF4 (K)およびMYCL (L)を加えた。尿路上皮細胞に関連した遺伝子と初期化遺伝子を組み合わせた幾つかの細胞では、UPK1bのmRNAが高いレベルで発現した。このことは、FOXA1が遺伝子の組み合わせに含まれることが必須であることを示唆した(図3)。次に、I、T及びHの遺伝子の必要性を確かめた。その結果、IではなくTまたはHが上皮細胞コロニー形成を促進させることがわかった(図4)。P、K及びLの必要性を、FTとの組み合わせにより決定した。FTLKの組み合わせは、UPK1bのmRNA発現と上皮細胞コロニーの形成を誘導した(図5A、B)。さらにFTLKを導入した細胞は、別の尿路上皮特異的マーカーであるウロプラキン2(UPK2)mRNAを発現した(図5A)。UPK1bは尿路上皮細胞以外にも角膜上皮や結膜上皮にも発現しているが、UPK2はより尿路上皮細胞に特異度が強いと報告されている(Habuka, M. et al., PLoS. One. 10, e0145301 (2015))。
F、T、L、Kが、UPK1bおよびUPK2の発現誘導に必要十分であるかどうかを検証した。UPK1bのmRNA発現量はF、L、Kで転換された細胞では上昇した一方、T、L、Kの組み合わせではUPK2のmRNA発現量は非常に高かった。L、Kの組み合わせ、及びF、L、Kの組み合わせでも有意にUPK2のmRNA発現量が増加した。F、T、L、Kの組み合わせは、UPK1bとUPK2両方のmRNA発現上昇を誘導した(図6A)。これらのことから、F、T、L、Kが、aHDFに尿路上皮細胞の形質を誘導する最適な遺伝子の組み合わせであると結論づけた。
培養プレートのコーティングが細胞のコンバート効率に与える影響を調べた。aHDFsの播種前に、培養プレートをコラーゲン、ポリ-L-リジン、あるいはラミニンでコーティングした。上皮細胞コロニーは、ラミニンでコーティングされた培養プレートでのみ形成され(図6B)、UPK1bとUPK2のmRNAの発現は最も高かった(図6C)。
F、T、L、Kを導入した細胞と導入しない細胞を、様々な培地で培養した。図6D、E及び図7に示されたように、F、T、L、Kで転換された細胞は、CnT-Prime培地で最も良く尿路上皮細胞コロニーを形成し、UPK1b、UPK2のmRNA発現量も顕著に高かった。尿路上皮細胞の培養に適していることが多く報告されている「Uromedium」培地は、上皮細胞コロニー形成だけでなく、線維芽細胞様細胞の増殖もサポートすることがわかった。これらの結果は、CnT-Prime培地が尿路上皮細胞様への転換に優れていることを示唆した。対照的に、CnT-Prime培地では、遺伝子導入を行っていない細胞の尿路上皮細胞へのコンバートは起きなかった。
ダイレクト・コンバートされた尿路上皮細胞(dUCs)の特徴。
実施例1の方法で転換された細胞は、ダイレクト・コンバートされた尿路上皮細胞(dUC)と表される。次に、細胞の詳細な特徴を調べた。経時的な解析では、F、T、L、Kで転換された細胞は、遺伝子導入8日後でUPK1bを顕著に発現し、発現は15日後にピークに達した(図8a)。UPK2のmRNA発現は、FTLKで転換された細胞で11日後から上昇し始め、21日後にピークに達した。上皮細胞コロニーの形成は、11日後に観察され、その後増加していった(図8b)。
UPK1bとUPK2の発現に加えて、一般的な上皮細胞で発現する膜貫通型タンパク質であるE-カドヘリンと、細胞骨格であるケラチン8/18の発現を確認した。免疫細胞化学的解析により、約42%の細胞がUPK1bを発現する一方、UPK2陽性、E-カドヘリン陽性、ケラチン8/12陽性細胞はそれぞれ23%、26%、26%であった(図8c、d)。この結果は、コンバージョン率が約25%であることを示唆した。
オズボーンらは、iPS細胞から内胚葉を経て尿路上皮細胞に分化させる方法を報告している(Osborn SL, et al., (2014) Stem Cell Transl Med 3(5):610-619.)。そこで、ダイレクト・コンバージョンで作成したdUCsと、オズボーンらの方法に多少の修正を入れて誘導したiPS細胞由来の尿路上皮細胞(iUCs)を、比較した(図9A)。dUCsはUPK1bとUPK2遺伝子の発現を強く発現した一方、iUCsは、UPK1aとUPK3aの遺伝子を強く発現した(図9B)。このことは、iUCsに比べてdUCsの方がより未成熟な尿路上皮細胞に似ていることを強く示唆した。
多能性、肝、小腸マーカーはdUCsでは発現しなかった。
線維芽細胞が一時的にiPS細胞または内胚葉性前駆細胞にコンバートされ、そこから尿路上皮細胞が生じた可能性を排除するため、コンバージョンの過程においてF、T、L、K導入細胞における多能性マーカーの発現と、非尿路上皮性の内胚葉マーカーの発現を調べた。結果、F、T、L、K導入細胞では、培養期間中、NANOG、POU5F1、LIN28、SOX2の遺伝子発現は観察されなかった。NANOGのタンパク質も同様に観察されなかった(図10)。同様に、F、T、L、K導入細胞は、小腸上皮細胞マーカーであるCDX2、および肝細胞マーカーであるアルブミン(ALB)のmRNAはどの観察時点においても発現しなかった(図11)。また、F、T、L、K導入細胞は、間葉系由来内皮細胞マーカーであるCD31及び外胚葉由来唾液腺のマーカーであるAQP5のmRNAを発現することもなかった(図11)。よって、F、T、L、K導入細胞は、dUCsへのコンバートの過程で多能性ステージを経ていないこと、および他の内胚葉由来の細胞にコンバートしなかったことを確認した。また間葉系や外胚葉系の細胞にもコンバートしなかったことを確認した。
dUCsは継代培養により選択的に増殖させることができる。
遺伝子導入21日後までに形成されたdUCコロニーは、同じ培地上でその後続けて培養されても活発に成長することはなかった。dUCsを高純度で大量に確保するために、細胞を最初の培地(世代数0、P0)で培養を開始し、その後新しい培地に継代した(世代数1、P1)。しかし、CnT-Prime培地で作られたP0のdUCsを同じ培地で新たに培養しても、上皮細胞コロニーはほとんど形成されず、上皮細胞ではない細胞が僅かに増殖するだけであった。「Uromedium」は、次の世代でも敷石様のコロニーを形成しなかった(図12)。そこで
、dUCsに適した培地にするために、「Uromedium」に含まれる培地成分の濃度を改良し、組成を決定した。図13Aに示したように、インスリンとヒドロコルチゾンの濃度は決定的でなかった。一方、F、L、T、K導入細胞がUPK2 mRNAを発現することも含めて、1-10 mMのIBMXが有益な役割を果たしていた。ヒドロコルチゾンを含まない培地と、10 mMのIBMXを含む培地では、F、L、T、K導入細胞が上皮細胞コロニーを形成した(図13B)。EGFは尿路上皮細胞の増殖、分化には必要されないと報告されていたが、0.01-1 ng/mLのEGFを加えることでUPK2のmRNAの発現は上昇した(図13A)。iPS細胞から線維芽細胞へのリプログラミング効率を増加させることで知られるモノアミン酸化酵素の非可逆的阻害剤であるトラニルシプロミンを添加することで、尿路上皮細胞コロニーの形成は促進した(図13C)。
これらの結果に基づき、dUCsの継代に最適な培地成分を決定した。尿路上皮細胞コンバージョン・維持培地(UCM)と名付けたこの培地は、以下の組成を有する。
60μMカルシウム、
60μg/mLウシ脳下垂体抽出液、
5μg/mLヒト遺伝子組み換えインスリン、
30μg/mLゲンタマイシン、
15ng/mLアンフォテリシン、
2%FBS、
0.01ng/mLのヒト遺伝子組み換えEGF、
1mMのIBMX、および
1μMのトラニルシプロミン
を添加したEpiLifeTM培地。
(EpiLifeTM培地:ケラチノサイト培養用の培地)
UCMは、F、L、T、K導入細胞の上皮細胞コロニー形成をサポートし、線維芽細胞様細胞の成長を抑制した(図12、図13D)。次に、この特徴的な培地を用いてdUCsの継代培養と選択的な成長がサポートされるのかを調べた(図14a)。図15aに示したように、少なくともdUCsの4世代継代培養に成功した。P4では、dUCsはUPK1b、UPK2およびE-カドヘリンを強く発現した(図15b,c及び図14b、c)。これらの結果は、UCM培地では、dUCsが線維芽様細胞よりも効率的に増殖し、継代の過程で安定的に尿路上皮様の細胞に留まっていたことを示唆する。
dUCsはインビトロにおいて顕著なバリア機能を発揮する。
次に、P4 dUCsのバリア機能を、トランスウェルを用いたインビトロ透過性アッセイを用いてチェックした。P4のdUCを、ラミニンでコーティングされたinner chamberの底部メンブレン上でコンフルエントになるまで培養した(図15d)。その後FITCで標識されたデキストラン(FITC-デキストラン)をinner chamber内に添加し、outer chamberへの漏れを検出した。細胞を入れないコントロール群およびaHDFsを培養したもう一つのコントロール群と比べると、FITC-デキストランの透過性はdUCsを培養したトランスウェルでは顕著に低かった(図15e)。HUCs及び最終分化したHUCsについてもまたFITC-デキストランの漏出は低下していた(図21)。このことは、本願発明の方法で作成されたP4 dUCsがインビトロにおいて上皮バリアを形成したことを示唆する。
RNAシーケンスにより、P2 dUCsの尿路上皮細胞としての特徴をさらに確認した。
RNAシーケンスにより、P2 dUCsと尿路上皮細胞の類似性を解析した。ゲノム全長の階層クラスタリングによる遺伝子発現プロファイリング解析により、dUCsはaHDFよりもHUCsにより類似していることが明らかになった(図16A)。図16Bは最も変動する50の遺伝子のデータを示す。この結果は、線維芽細胞ではほとんど発現していないケラチン(KRT8、18、5、17、6A、14)とウロプラキンの細胞上部への輸送に関わるRAB27Bを含む尿路上皮細胞に関連した遺伝子を、dUCsとHUCsはどちらも特徴的に発現することを示している。一方で、線維芽細胞に関連した遺伝子(FAP、COL16A1、COL1A2、DPT、PTX3他)は、dUCsとHUCsにおいて発現が減少していた(図16B)。遺伝子発現解析はさらに、dUCsが線維芽細胞ではなく尿路上皮細胞に関連した遺伝子を強く発現することをより確かなものとした(図16C、図17A)。ケラチンフィラメント及びコラーゲン線維組織に関連した遺伝子に関しては、HUCs及びdUCsは同様の発現パターンを示した(図17A)。特筆すべきは、dUCsが膀胱上皮細胞の発生に関わる重要な因子の一つであるKLF5を発現したことである(図16C)。dUCsはまた、KEGGパスウェイ解析により、上皮間の細胞間接着タイトジャンクション形成に関わる遺伝子を強く発現したことがわかった(図17B)。これらの遺伝子も線維芽細胞ではほとんど発現していない。
dUCsはインビボにおいて損傷した尿路上皮細胞の再生に寄与する可能性がある。
F、T、L、Kを導入したaHDFsがインビボにおいてdUCsにコンバートし、尿路上皮細胞の再生に寄与するかどうかを調べた。F、T、L、KおよびGFP遺伝子を導入されたaHDFsは、CnT-Prime培地で21日間培養すると、GFPでラベルされたdUCsへとコンバートした(図18a、b)。インビボの実験では、aHDFsをF、T、L、K、G遺伝子導入4日後に間質性膀胱炎で損傷したマウスの膀胱に移植した。移植後1週間で、GFPで標識した細胞は、膀胱の粘膜上皮内に存在し、UPK1b、UPK2、E-カドヘリン、ケラチン8/18を発現した(図18d)。また、最終分化したアンブレラ細胞において発現するとされているKRT20も発現していた。対照的に、GFPで標識したaHDFsを膀胱内に移植しても、GFP陽性かつE-カドヘリン陽性の細胞は観察されなかった(図18d)。よって、F、L、T、K、Gを導入したaHDFは、in vivoでdUCsにコンバートされ、損傷した膀胱の上皮に局在して組織の再生に関わっていた。
dUCsの造腫瘍性の有無の検討
dUCsの潜在的な造腫瘍性を調べた。これまでにテストしたところ、F、L、T、Kを導入したaHDFsは、免疫不全マウスの皮下に接種された後も腫瘍を形成しなかった(図19)。一方T24膀胱がん細胞は腫瘍を形成した。
実施例1と同様にして、aHDFsに、特定の因子を導入することにより尿路上皮細胞様の表現型を発現することを確認した。結果を図20に示す。F、T、L、K又はそれらの組み合わせが、UPK1bおよびUPK2の発現誘導に有効であることが示された。
本出願は、日本で出願された特願2019-159070(出願日:2019年8月30日)を基礎としておりその内容は本明細書に全て包含されるものである。
Claims (20)
- 哺乳動物の体細胞に、外来性因子として、
FOXA1(Forkhead box A1)遺伝子若しくはその発現産物、
TP63(tumor protein P63)遺伝子若しくはその発現産物、
MYCL(L-Myc)遺伝子若しくはその発現産物、及び
KLF4(Kruppel-like factor 4)遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を導入する工程を含む、尿路上皮細胞を誘導する方法。 - 外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、請求項1記載の方法。
- 外来性因子が、以下より選択される、請求項1記載の方法;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。 - 前記体細胞がヒト線維芽細胞である、請求項1~3のいずれか1項に記載の方法。
- 哺乳動物の体細胞に由来し、外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を有する、尿路上皮細胞。 - 外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、請求項5記載の細胞。
- 外来性因子が、以下より選択される、請求項5記載の細胞;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。 - 前記体細胞がヒト線維芽細胞である、請求項5~7のいずれか1項に記載の細胞。
- 外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を含む、哺乳動物の体細胞を尿路上皮細胞にコンバートする為の剤。 - 外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、請求項9記載の剤。
- 外来性因子が、以下より選択される、請求項9記載の剤;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。 - 前記体細胞がヒト線維芽細胞である、請求項9~11のいずれか1項に記載の剤。
- 外来性因子として、
FOXA1遺伝子若しくはその発現産物、
TP63遺伝子若しくはその発現産物、
MYCL遺伝子若しくはその発現産物、及び
KLF4遺伝子若しくはその発現産物
からなる群より選択される少なくとも1種を含む、哺乳動物の体細胞を尿路上皮細胞にコンバートする為のベクター。 - 外来性因子が、FOXA1遺伝子若しくはその発現産物、及びTP63遺伝子若しくはその発現産物を含む、請求項13記載のベクター。
- 外来性因子が、以下より選択される、請求項13記載のベクター;
(i) KLF4遺伝子若しくはその発現産物
(ii) TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iii) MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(iv) FOXA1遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(v) TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vi) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物
(vii) FOXA1遺伝子若しくはその発現産物、TP63遺伝子若しくはその発現産物、MYCL遺伝子若しくはその発現産物、及びKLF4遺伝子若しくはその発現産物。 - 前記体細胞がヒト線維芽細胞である、請求項13~15のいずれか1項に記載のベクター。
- 請求項1~4のいずれか1項に記載の方法で得られる細胞、請求項5~8のいずれか1項に記載の尿路上皮細胞、請求項9~12のいずれか1項に記載の剤、又は請求項13~16のいずれか1項に記載のベクターを含む、泌尿器系の疾患の治療剤。
- 泌尿器系の疾患が、尿路疾患である、請求項17に記載の治療剤。
- 尿路疾患が、膀胱がん、過活動膀胱、膀胱の先天異常、膀胱損傷、間質性膀胱炎、神経因性膀胱及び萎縮膀胱からなる群より選択される少なくとも1種である、請求項18に記載の治療剤。
- 3-イソブチル 1-メチルキサンチン(IBMX)及び上皮成長因子(EGF)を含むことを特徴とする、尿路上皮細胞誘導用培地。
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EP4023248A4 (en) | 2023-10-11 |
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