WO2011155641A1

WO2011155641A1 - Method for producing erythropoietin, and method for isolating erythropoietin-producing cells

Info

Publication number: WO2011155641A1
Application number: PCT/JP2011/063861
Authority: WO
Inventors: Motoko Yanagita; Nariaki Asada
Original assignee: Kyoto University NUC
Current assignee: Kyoto University NUC
Priority date: 2010-06-10
Filing date: 2011-06-10
Publication date: 2011-12-15
Anticipated expiration: 2012-12-10
Also published as: JP2013116854A

Abstract

The present invention provides a method for producing erythropoietin comprising culturing neural crest-derived cells in a medium to allow production of erythropoietin and recovering erythropoietin from the medium. The present invention also provides a method for isolating erythropoietin-producing cells from a renal tissue comprising isolating erythropoietin-producing cells from a cell population contained in the renal tissue using a neural crest-derived cell-specific surface antigen(s).

Description

DESCRIPTION

METHOD FOR PRODUCING ERYTHROPOIETIN, AND METHOD FOR ISOLATING ERYTHROPOIETIN-PRODUCING CELLS

TECHNICAL FIELD

[0001]

The present invention relates to a method for producing erythropoietin, method for isolating erythropoietin-producing cells, and a pharmaceutical prepared using the erythropoietin-producing cells.

BACKGROUND ART

[0002]

Erythropoietin (EPO) is a hormone essential for the production of red blood cells. EPO is mainly produced in the kidney, and its production is severely reduced in patients suffering from chronic kidney disease. That is, in patients suffering from chronic renal failure, production of EPO from the kidney is reduced, leading to renal anemia. In recent years, recombinant EPO has been used for the treatment of chronic renal failure, and the medical expenses therefor reach 140 billion yen per year. However, recombinant EPO is synthesized using non-human cells; their preparation requires much labor and cost; and recombinant EPO is differently modified as compared to endogenous human EPO; so that recombinant EPO has lower activities and stabilities than the endogenous EPO, and hence their repeated administration is necessary. Since, in clinical sites, administration of a recombinant EPO is carried out once a week by subcutaneous injection, the changes in its blood level lack the circadian rhythm, which is observed for the endogenous EPO, and also lack responses to secretory stimuli such as hypoxia and inflammation.

[0003] Although the physiological roles of EPO have been analyzed in detail, the properties of EPO-producing cells in the kidney have not been revealed yet. It has been shown by in situ hybridization that EPO and ecto-5' -nucleotidase are

colocalized around renal tubules in the renal cortex (J Histochem Cytochem 41, 335- 41 (1993)). Further, it has been reported, based on the results of analysis using a transgenic mouse that expresses EGFP under the control of the EPO promoter, that EPO is produced mainly in interstitial "fibroblast-like" cells having characteristics of nerve cells in the cortex and the surface of the outer medulla of the kidney (Blood 111, 5223-32 (2008)), but most of the EPO-producing cells cannot be explained by the interstitial "fibroblast-like" cells.

Further, many studies have been carried out based on the idea that EPO- producing cells are generated from stem cells, and, in Transplantation 85(11), 1654- 1658 (2008), a method of induction of EPO-producing cells by culturing

mesenchymal stem cells on the lesser omentum of the rat stomach has been disclosed. There are also attempts to induce ES cells and iPS cells into the direction of the kidney to prepare EPO-producing cells, but these have not been successful so far.

[0004]

Further, in World J Urol. 26(4), 295-300 (2008), it has been reported that EPO-producing cells were isolated from the mouse kidney using an anti-EPO antibody. In this document, it is described that all the cells obtained by mincing the renal cortex were plated in a culture dish and EPO-producing cells were then obtained using an anti-EPO antibody, but there is no evidence at all which supports the fact that EPO-producing cells are concentrated by this method. After all, since EPO is a protein existing inside the cell when it is produced, it cannot be detected by flow cytometry without forming a hole on the cell membrane to allow the antibody to reach the inside of the cell, but, in this case, the cell is killed, so that it is impossible to obtain the cells by sorting using an anti-EPO antibody and culture the cells. Further, in this document, the anti-EPO antibody employed has not been evaluated, and hence the results are very unreliable. Actually, efficient isolation of EPO- producing cells has not been achieved so far, and the properties and behaviors of EPO-producing cells have not been sufficiently clarified yet.

[0005]

The neural crest is a transient tissue that originates from the dorsal region of the neural tube during vertebrate development (J Cell Biochem 107, 1046-52 (2009)). Neural crest-derived cells delaminate from the neural tube and migrate to various directions, where they differentiate into a vast array of cell types including the peripheral nervous system and craniofacial skeleton. Neural crest cells also give rise to endocrine cells such as chromaffin cells of the adrenal medulla, chief cells of extra-adrenal paraganglia, and thyroid endocrine cells (C R Biol 330, 521-9 (2007)). There is also a hypothesis suggesting that interstitial stromal cells of kidneys during differentiation are also of neural crest origin, and it has been reported, by a

transplantation experiment at an early stage, that transplantation of the quail neural crest to a chicken embryo resulted in appearance of quail neural crest cells in the kidney interstitium of the chicken embryo (Dev Biol 41, 162-84 (1974); Nature 335, 161-4 (1988)). Further, there is also a report suggesting that developing renal interstitial cells express a disialylated ganglioside GD3 and the neurofilament light chain and medium chain proteins, which are neural crest markers (Cell 54, 235-45 (1988); Int J Dev Biol 38, 77-84 (1994)). However, there has been no report suggesting that neural crest-derived cells exist in the adult kidney and are functioning physiologically.

SUMMARY OF THE INVENTION

[0006]

The present invention aims to provide a method of efficiently producing EPO having similar properties to naturally occurring EPO. The present invention also aims to provide a method of efficiently isolating EPO-producing cells, and to provide a pharmaceutical prepared using the obtained EPO-producing cells.

[0007]

The present inventors intensively studied to solve the above-described problems. As a result, the present inventors discovered that neural crest-derived cells in the kidney produce EPO, and that EPO can be efficiently produced by isolating the neural crest cells using their surface antigen(s) or inducing neural crest cells from iPS cells or the like, followed by culturing the obtained cells under appropriate conditions, thereby completed the present invention.

[0008]

That is, the present invention provides the followings.

[1] A method for producing erythropoietin, said method comprising culturing neural crest-derived cells in a medium to allow production of erythropoietin and recovering erythropoietin from the medium.

[2] The method according to [1], wherein said neural crest-derived cells are induced from pluripotent stem cells.

[3] A method for isolating erythropoietin-producing cells from a renal tissue, said method comprising isolating erythropoietin-producing cells from a cell population contained in the renal tissue using a neural crest-derived cell-specific surface antigen(s).

[4] The method according to [3], wherein said neural crest-derived cell-specific surface antigen(s) is/are one or more selected from the group consisting of p75, PDGFR (platelet-derived growth factor receptor) a and PDGFRp.

[5] A method for culturing erythropoietin-producing neural crest cells, said method comprising culturing erythropoietin-producing neural crest cells in a medium containing one or more factor(s) selected from the group consisting of PDGF, NGF, HGF, Tsukushi, Activin, BMP-7, LIF, EGF family ligands, estrogen, androgen, BDNF, GDNF, retinoic acid, bFGF, jagged- 1, TGFa, and angiotensin II.

[6] A graft material for treatment of a renal disease, said graft material

comprising neural crest-derived cells.

[7] A growth-promoting agent for erythropoietin-producing cells, said growth- promoting agent comprising one or more factor(s) selected from the group consisting of PDGF, NGF, HGF, Tsukushi, Activin, BMP-7, LIF, EGF family ligands, estrogen, androgen, BDNF, GDNF, retinoic acid, bFGF, jagged- 1, TGFa, and angiotensin II.

[8] A method of treating a renal disease, comprising grafting neural crest- derived cells into a subject in need of treatment of the renal disease.

[9] Neural crest-derived cells, for use in treatment of a renal disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

Fig. 1 shows micrographs showing the results of EGFP immunostaining of the mouse kidney. (A) PO-Cre/Floxed-EGFP mouse (low-power field); (B) P0- Cre/Floxed-EGFP mouse (high-power field); (C) control mouse (P0-Cre mouse which does not have the CAG-EGFP allele).

Fig. 2 shows micrographs showing the results of EGFP/marker double staining of the kidney of a PO-Cre/Floxed-EGFP mouse. (A) CD73/5 'NT (ecto-5 ' - nucleotidase) and EGFP; (B) PDGF receptor β (PDGFR β) and EGFP; (C) aSMA and EGFP.

Fig. 3 shows micrographs showing comparison between the result of lacZ staining of a P0-Cre/R26R mouse and the result of EGFP/marker double staining of a PO-Cre/Floxed-EGFP mouse. (A) LacZ staining of the kidney of a P0-Cre/R26R mouse; (B) p75/EGFP staining of the kidney of a PO-Cre/Floxed-EGFP mouse; (C) PDGFRa/EGFP staining of the kidney of a PO-Cre/Floxed-EGFP mouse; (D) Thyl/EGFP staining of the kidney of a PO-Cre/Floxed-EGFP mouse.

Fig. 4 shows micrographs showing the results of lacZ staining of the kidney of a P0-Cre/R26R mouse on embryonic day 13.5. (A) Low-power field; (B) high- power field; (C) Six2 staining; (D) p75 staining; (E) PDGFRa staining.

Fig. 5 shows micrographs showing the results of lacZ and EGFP staining of the kidney of a P0-Cre/R26R/EPO-EGFP mouse. (A) LacZ; (B) EGFP; (C) superimposed image.

Fig. 6 shows diagrams (photographs) showing the results of RT-PCR for investigating EPO expression in the kidney of a P0-Cre/R26R/EPO-EGFP mouse. (A) Cells were sorted from the mouse kidney using an anti-EGFP antibody, and RT- PCR was carried out for EPO, p75 and GAPDH using the obtained cells. (B) Cells were sorted from the mouse kidney using an anti-p75 antibody, and RT-PCR was carried out for EPO, EGFP and GAPDH using the obtained cells.

Fig. 7 shows micrographs showing the effects of various cytokines on the growth of isolated neural crest-derived cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010]

The method of the present invention for producing erythropoietin comprises culturing neural crest-derived cells in a medium to allow production of erythropoietin and recovering erythropoietin from the medium.

Here, the neural crest-derived cells are preferably fibroblasts.

The neural crest is a transient tissue that appears, when the neural tube is formed during vertebrate development, on the border between the neuroectoderm and the epidermal ectoderm, and the cell population that deepithelializes and migrates therefrom is called neural crest cells (J Cell Biochem 107, 1046-52 (2009)).

[0011] Neural crest-derived cells can be defined by existence of cell surface markers such as p75, PDGFR (platelet-derived growth factor receptor) a and PDFGRp, which are neural crest markers ( J Cell Biochem 107, 1046-52 (2009); Stem Cells Dev. 2009, 18(7):1059-1070; Development 1997 124(14) p2691-2700).

[0012]

The medium to be used for the culture of neural crest-derived cells may be one conventionally used for culture of fibroblasts, and examples thereof include, but are not limited to, minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, 199 medium, F12 medium and DMEM/F12 medium, which are supplemented with about 5 to 20% fetal bovine serum.

[0013]

To the medium for culturing the neural crest-derived cells, a factor(s) for promotion of the growth of EPO-producing cells is/are preferably added. Examples of such a component(s) include the followings. These factors are preferably added to a concentration of 10 to 100 ng/ml.

PDGF (platelet-derived growth factor: PDGF-AA, PDGF-AB, PDGF-BB)

NGF (nerve growth factor)

HGF (hepatocyte growth factor)

Tsukushi (Development. 2006 Jan; 133(l):75-88)

Activin

BMP (Bone Morphogenetic Protein)-7

LIF (Leukemia Inhibitory Factor)

EGF (Epidermal Growth Factor) family ligands (EGF, amphiregulin, HB- EGF, Nrg-1)

Estrogen

Androgen

BDNF (Brain-derived neurotrophic factor) GDNF (Glial cell-line derived neurotrophic factor)

Retinoic acid

bFGF (basic fibroblast growth factor)

jagged- 1

TGF (Transforming growth factor) a

Angiotensin II

[0014]

Since these substances can promote the growth of EPO-producing cells or production of EPO by these cells, the substances may be used as growth-promoting agents for erythropoietin-producing cells.

[0015]

The culture may be carried out under conventional conditions for cell culture, for example, at 37°C under 5% C0₂.

[0016]

The EPO secreted into the medium by the culture can be purified according to a known method. For example, EP 0267678 A discloses purification of EPO produced by serum-free culture, which comprises dialysis, ion-exchange

chromatography on S-Sepharose, preparative reverse-phase HPLC on a C8 column and gel filtration chromatography.

Further, Nobuo, I. et al. (J. Biochem. 107:352-359 (1990)) describes a process of purification of a recombinant EPO, and similar methods may also be employed. Further, EPO may also be purified by affinity purification using an anti-EPO antibody.

[0017]

The neural crest-derived cells to be used for the culture may be those isolated from the kidney or bone marrow of a mammal.

The neural crest-derived cells can be obtained by isolating the kidney from a small mammal such as mouse or rat; or a large mammal such as pig or cow; to obtain a cell population of the kidney, and sorting the cells by a method such as flow cytometry or the like using the above-described neural crest marker (cell surface antigen)-specific antibody.

Further, from human kidney tissues obtained by renal biopsy or surgery, neural crest-derived cells may be isolated, to be used for the culture.

[0018]

To obtain a large amount of neural crest-derived cells efficiently, the neural crest-derived cells are preferably induced from embryonic stem cells (ES cells) or pluripotent stem cells such as induced pluripotent stem cells (iPS cells).

The method for producing the iPS cells is described below.

The somatic cells to be used as starting materials for preparation of the iPS cells may be any cells other than germ cells, and examples of the somatic cells include epithelial cells which are keratinized (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the lingual surface), epithelial cells of exocrine glands (e.g., mammary cells), hormone-secreting cells (e.g.,

adrenomeduUary cells), cells for metabolism and storage (e.g., hepatic cells), luminal epithelial cells constituting boundary surfaces (e.g., type I alveolar cells), luminal epithelial cells in the closed circulatory system (e.g., vascular endothelial cells), ciliated cells having a carrying capacity (e.g., tracheal epithelial cells), extracellular matrix-secreting cells (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), cells involved in the blood system and the immune system (e.g., T lymphocytes), sensory cells (e.g., rod cells), autonomic neurons (e.g., cholinergic neurons), supporting cells of sense organs and peripheral neurons (e.g., satellite cells), nerve cells and glial cells in the central nervous system (e.g., astroglial cells) and pigment cells (e.g., retinal pigment epithelial cells), and progenitor cells (tissue progenitor cells) thereof. By introducing nuclear reprogramming factors to such somatic cells, iPS cells can be obtained. The nuclear reprogramming factors may be the genes described in WO 2007/069666. More particular examples of the nuclear reprogramming factors include Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c- Myc, L-Myc, N-Myc, TERT, S V40 Large T antigen, HPV 16 E6, HPV 16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, Esrrg and combinations thereof. Among the combinations of these factors, examples of preferred nuclear reprogramming factors include the combination of the 4 factors Oct3/4, Sox2, Klf4 and c-Myc, and the combination of the 3 factors Oct3/4, Sox2, and Klf4. The sequence information of human cDNAs of the respective nuclear reprogramming factors described above can be obtained by referring to the NCBI accession numbers described in WO

2007/069666, and those skilled in the art can easily isolate these cDNAs.

[0019]

The induction of neural crest cells from iPS cells is described in Nat

Biotechnol 27, 275-80 (2009).

That is, by culturing the obtained iPS cells in the presence of Noggin and SB431542, neural crest-derived cells are induced. However, the method of induction of neural crest-derived cells from iPS cells is not restricted thereto.

[0020]

Encapsulation of the neural crest-derived cells in a biocompatible polymer followed by transplantation (graft) of the encapsulated cells to patients suffering from renal failure, renal anemia or the like is useful for therapy for supplementing the EPO production of the patients suffering from the renal diseases. The site of transplantation is not restricted as long as the therapeutic effect can be attained, and subcutaneous transplantation is preferred in view of simplicity of the transplantation.

[0021]

The cell encapsulation method is known in the art, and disclosed in the following references.

J. Mol. Med. 77:199-205 (1999),

Adv. Drug Del Rev. 42:29-64 (2000).

US Patent No. 5,762,959

US Patent No.5,550,178

US Patent No.5,578,314

WO 91/07951

US Patent No. 4,353,888

[0022]

Further examples of proposed methods of encapsulation of cells include a microcapsule formed by the ion-ion interaction between acrylic polymers (JP 5- 34946 B), collagen gel-containing capsule (JP 63-3786 A), polyionic

microencapsulation using a cationic polysaccharide (JP 60-75326 A), and a capsule prepared by using an agarose decomposition product (JP 62-215530 A).

EXAMPLES

[0023]

The present invention will now be described more specifically by referring to Examples. However, the present invention is not restricted to the Examples below.

[0024]

To investigate the distribution of neural crest-derived cells in the kidney, kidneys of a PO-Cre/Floxed-EGFP mouse (Stem Cells. 2006 Dec; 24(12):2714-22) and a P0-Cre/R26R mouse (Circ Res. 2006 Jun 23; 98(12): 1547-54) were histologically analyzed. These mice were obtained by mating a mouse that expresses Cre under the control of the P0 promoter with a CAG-CAT^loxP/loxP-EGFP mouse or a R26R mouse (mouse having a lacZ cassette incorporated in the ROSA region). P0 (Protein 0) is expressed in the migrating neural crest cells at the early embryonic stage and Schwann cells. In these transgenic mice, activation of the P0 promoter in migrating neural crest cells promotes recombination by Cre, which allows tagging of neural crest cells by expression of indicator genes.

[0025]

Immunostaining of the kidney was carried out as follows. The isolated kidney was fixed with 4% paraformaldehyde, and embedded in Cryomold

(trademark), followed by slicing the resultant into 6-μηι sections. As a primary antibody, an anti-EGFP antibody (Molecular Probe), anti-p75 antibody (Advanced Targeting Systems), anti-TH antibody (Chemicon), anti-Cre antibody (Novagen) or anti-PO antibody (Aves) was used. As a secondary antibody, fluorescently labeled Alexa 488 or Alexa 568 (Molecular Probes) was used.

[0026]

As a result of EGFP immunostaining of the kidney of the P0-Cre/Floxed-

EGFP mouse (8 weeks old), it was revealed that many EGFP-positive cells exist in the cortex and the outer medulla (Fig. 1 A and B). On the other hand, in EGFP immunostaining of the kidney of the P0-Cre mouse, which is a control having no floxed-EGFP, EGFP-positive cells were not detected (Fig. 1C).

The result of LacZ staining of the kidney of the P0-Cre/R26R mouse was almost the same as the result for the PO-Cre/Floxed-EGFP mouse.

These cells existed in the interstitium, that is, the space between tubules and nutritive peritubular capillaries (PTCs), and displayed a unique stellar shape with numerous projections extending to various directions. From these observation results, it was suggested that neural crest-derived cells migrate into the kidney interstitium, where they survive.

[0027] Further, the result of double staining of the kidney of the PO-Cre/Floxed- EGFP mouse (8 weeks old) with EGFP and the fibroblast marker CD73/5'NT (ecto- 5 '-nucleotidase) or PDGF receptor β (PDGFR-β) demonstrated expression of these markers by EGFP-positive cells (Fig. 2A and 2B). Fibroblasts of the neonatal kidney are reported to express, in addition to these well-known markers, aSMA, which is a marker of myofibroblasts in the diseased kidney of adults (Am J Pathol 173, 1617-27 (2008)). As a result of double staining with EGFP and aSMA, it was suggested that most of the EGFP-positive cells in the neonatal kidney express aSMA (Fig. 2C), and hence the EGFP-positive cells in the kidney of the PO-Cre/Floxed- EGFP mouse are fibroblasts.

[0028]

Further, as a result of staining of the kidney of the P0-Cre/R26R mouse (8 weeks old) and the PO-Cre/Floxed-EGFP mouse (8 weeks old), it was confirmed that these EGFP-positive fibroblasts and LacZ-positive fibroblasts express the neural crest markers p75 and PDGFRa, as well as Thy 1 , which is a marker of fibroblasts derived from neural crest stem cells (Fig. 3 A to D). From these results, it was revealed that most, although not all, of the fibroblasts existing in the cortex and the outer medulla of the kidney are of neural crest origin, and still express neural crest markers.

[0029]

As described above, neural crest-derived cells differentiate into fibroblasts in the adult and neonatal kidney interstitium. In order to investigate the migration pathway of neural crest cells to the kidney, histological analysis was carried out for P0-Cre/R26R mouse embryos serially from embryonic day 11.5, when nephrogenesis is initiated, to the neonatal stage. LacZ⁺ cells were hardly observed until embryonic day 12.5, but, from embryonic day 13.5, many of them were observed in the metanephric kidneys, especially along the outer capsule and the ureter of the kidney, indicative of possible routes of migration (Fig. 4A and B). The LacZ⁺ cells along the outer capsule were considered to be migrating to the cortex avoiding Six2⁺ metanephric mesenchymal cells (Fig. 4C).

[0030]

Thereafter, the signaling pathway that induces migration of neural crest- derived cells to the metanephric kidneys was analyzed. Expression of the neural crest markers p75 and PDGFRa by LacZ⁺ cells was observed (Fig. 4D and E).

Neural crest cells that express PDGFRa migrate to organs expressing the ligands PDGF-A and PDGF-C (Genes Dev 22, 1276-312 (2008)). Li et al. reported that PDGF-A and PDGF-C are strongly expressed in early-stage renal epithelial aggregates and the metanephric interstitium (Nat Cell Biol 2, 302-9 (2000)).

[0031]

Thereafter, production of EPO by neural crest-derived fibroblasts existing in the kidney was investigated. First, a P0-Cre/R26R mouse was mated with an EPO- EGFP mouse (transgenic mouse that expresses EGFP under the control of the promoter of the mouse Epo gene of 180 kb). As a result of analysis using the obtained mouse, fluorescence of EGFP was detected in the kidney after induction of anemia, and therefore existence of EPO-producing cells was clarified. Analysis of the kidney of the P0-Cre/R26R/EPO-EGFP mouse showed that about 70% of the EGFP⁺ cells are positive for LacZ (Fig. 5 A to C), and hence that neural crest-derived cells differentiate into EPO-producing fibroblasts.

[0032]

To confirm this experimental result, the kidney of a PO-Cre/Floxed-EGFP mouse was taken, and EGFP⁺ cells were sorted with FACSAria. More particularly, mice were anesthetized, and perfused with physiological saline, followed by extirpation of the kidney, mincing of the extirpated kidney with a razor, and treatment with collagenase. The obtained cell suspension was treated with

CellStrainer (trademark) to remove debris, and cells reactive to the anti-EGFP antibody were obtained.

From the obtained cells, RNA was extracted, and RT-PCR was carried out using the RNA. As a result, high-level expressions of EPO and p75 could be confirmed in EGFP⁺ cells, but their expression was not observed in EGFP^" cells (Fig. 6A).

Further, similarly, using the anti-p75 antibody, p75⁺ cells were sorted from the kidney of a PO-Cre/Floxed-EGFP mouse. The obtained p75⁺ cells were observed for expression of EPO and EGFP, and, as a result, high-level expressions of EPO and p75 were observed in p75⁺ cells, but their expression was not observed in p75^" cells (Fig. 6B). From these results, it was shown that EPO-producing cells are of neural crest origin. The primers used for the RT-PCR were designed using Primer Express software (Applied Biosystems), and the reaction was carried out at 50°C for 2 minutes and then at 95°C for 10 minutes, followed by 40 cycles of (95°C for 15 seconds and 60°C for 1 minute).

[0033]

Thereafter, the effects of various cytokines on the growth of the isolated neural crest-derived cells were investigated. More particularly, 1 ^χΐθ⁵ neural crest- derived cells isolated from the kidney of a mouse using EGFP were suspended in 2 ml of DMEM/10% FCS medium (cytokine-free), and the cells were cultured at 37°C under 5% C0₂ for 72 hours, to investigate their growth. The results are shown in Fig. 7. As a result, it was revealed that growth of EPO-producing cells is promoted upon stimulation with HGF or NGF. INDUSTRIAL APPLICABILITY

[0034]

By the method of the present invention, EPO having a high activity and a high stability, which could not be obtained with conventional recombinant techniques, can be produced efficiently. In particular, use of neural crest cells induced from iPS cells even allows mass production of such EPO. EPO is effective for treatment of chronic renal diseases and the like. Further, since EPO is suggested to also have a protective role against various nonhematopoietic tissues, it may be also applicable to tissue disorders in the future.

Further, by encapsulating the isolated EPO-producing cells and transplanting the encapsulated cells, tumorigenesis of the transplanted cells and immune response against the transplanted cells can be avoided, while achieving production and secretion of EPO depending on the conditions in the body.

Claims

1. A method for producing erythropoietin, said method comprising culturing neural crest-derived cells in a medium to allow production of erythropoietin and recovering erythropoietin from the medium.

2. The method according to claim 1, wherein said neural crest-derived cells are induced from pluripotent stem cells.

3. A method for isolating erythropoietin-producing cells from a renal tissue, said method comprising isolating erythropoietin-producing cells from a cell population contained in the renal tissue using a neural crest-derived cell-specific surface antigen(s).

4. The method according to claim 3, wherein said neural crest-derived cell- specific surface antigen(s) is/are one or more selected from the group consisting of p75, PDGFR (platelet-derived growth factor receptor) a and PDGFRp.

5. A method for culturing erythropoietin-producing neural crest cells, said method comprising culturing erythropoietin-producing neural crest cells in a medium containing one or more factor(s) selected from the group consisting of PDGF, NGF, HGF, Tsukushi, Activin, BMP-7, LIF, EGF family ligands, estrogen, androgen, BDNF, GDNF, retinoic acid, bFGF, jagged- 1, TGFa, and angiotensin II.

6. A graft material for treatment of a renal disease, said graft material

comprising neural crest-derived cells.

7. A growth-promoting agent for erythropoietin-producing cells, said growth- promoting agent comprising one or more factor(s) selected from the group consisting of PDGF, NGF, HGF, Tsukushi, Activin, BMP-7, LIF, EGF family ligands, estrogen, androgen, BDNF, GDNF, retinoic acid, bFGF, jagged- 1, TGFa, and angiotensin II.

8. A method of treating a renal disease, comprising grafting neural crest- derived cells into a subject in need of treatment of the renal disease.

9. Neural crest-derived cells, for use in treatment of a renal disease.