CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of International Patent Application No. PCT/JP2015/080016, filed on Oct. 23, 2015, which claims the benefit of Japanese Patent Application No. 2014-217867, filed on Oct. 24, 2014, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

TECHNICAL FIELD

The present invention relates to a method for producing neural tissues such as retinal tissue and the like from pluripotent stem cells.

BACKGROUND ART

As a method for producing neural tissues such as retinal tissue and the like from pluripotent stem cells, a method for producing neural tissue which comprises forming uniformed aggregates of pluripotent stem cells in a serum-free medium, culturing them in suspension, culturing them in suspension in a culture medium for differentiation induction in the presence of a differentiation-inducing factor and the like as appropriate to induce differentiation of pluripotent stem cells into the intended neural cells has been reported (patent document 1 and non-patent document 1). For example, a method for obtaining a multi-layered retinal tissue from pluripotent stem cells (non-patent document 2 and patent document 2), and a method for obtaining multi-layered retinal tissue which comprises forming uniformed aggregates of pluripotent stem cells in a serum-free medium containing a Wnt signal transduction pathway inhibiting substance, culturing them in suspension in the presence of a basement membrane preparation, and culturing them in suspension in a serum-containing medium (non-patent document 3 and patent document 3) are known. In addition, a method for inducing differentiation of pluripotent stem cells into a hypothalamic tissue (patent document 4 and non-patent document 4), and a method inducing differentiation of pluripotent stem cells into neural precursor cells (non-patent document 5 and 6) have also been reported.

The pluripotent stem cells as a starting material of these production methods, particularly in the case of primate pluripotent stem cells, were cultured to maintain undifferentiated state in the presence of feeder cells and with the addition of a factor for maintaining undifferentiated state. In recent years, improvement has been made in the culturing to maintain undifferentiated state, and a method of culturing primate pluripotent stem cells in the absence of feeder cells (feeder-free) with the addition of a factor for maintaining undifferentiated state has been reported (non-patent documents 7, 8 and 9). A stable method for producing neural cells or a neural tissue, which uses pluripotent stem cells subjected to feeder-free culture by this method as a starting material has been desired.

DOCUMENT LIST

Patent Documents

[patent document 1] WO 2009/148170[patent document 2] WO 2011/055855[patent document 3] WO 2013/077425[patent document 4] WO 2013/065763

Non-Patent Documents

[non-patent document 1] Cell Stem Cell, 3, 519-32 (2008)[non-patent document 2] Nature, 472, 51-56 (2011)[non-patent document 3] Cell Stem Cell, 10(6), 771-775 (2012)[non-patent document 4] Nature, 480, 57-62 (2011)[non-patent document 5] Nature Biotechnology, 27(3), 275-80 (2009)[non-patent document 6] Proc Natl Acad Sci USA, 110(50), 20284-9 (2013)[non-patent document 7] Nature Methods, 8, 424-429 (2011)[non-patent document 8] Scientific Reports, 4, 3594 (2014)[non-patent document 9] In Vitro Cell Dev Biol Anim., 46, 247-58 (2010)

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

The problem to be solved by the present invention is provision of a method for producing neural tissues such as retinal tissue and the like and neural cells from pluripotent stem cells prepared or cultured to maintain undifferentiated state in the absence of feeder cells.

Means of Solving the Problems

The present inventors have conducted intensive studies in an attempt to solve the aforementioned problems and found that a spherical cell aggregate having a smooth surface and a dense inside, and maintaining an undifferentiated state can be formed with high efficiency by culturing pluripotent stem cells in the absence of feeder cells (under feeder-free condition) in a medium containing a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and then subjecting the cells to suspension culturing. In addition, the present inventors have found that by using this high quality cell aggregate, neural tissues such as retinal tissue and the like and neural cells can be induced with high efficiency, which resulted in the completion of the present invention.

That is, the present invention relates to the following.

[1] A method for producing neural cells or a neural tissue, comprising the following steps (1)-(3):

(1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising 1) a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state,
(2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and
(3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a differentiation-inducing factor to obtain an aggregate containing neural cells or a neural tissue.
[2] The production method of [1], wherein, in the second step, the cells obtained in the first step are dispersed, and the dispersed cells are cultured in suspension.
[3] The production method of [1] or [2], wherein the factor for maintaining undifferentiated state is an FGF signal transduction pathway activating substance.
[4] The production method of [3], wherein the FGF signal transduction pathway activating substance is bFGF.
[5] The production method of any of [1]-[4], wherein, in the second step, the cells are cultured in suspension in a serum-free medium containing a Sonic hedgehog signal transduction pathway activating substance.
[6] The production method of any of [1]-[5], wherein, in the third step, the aggregate is cultured in suspension in the presence of a differentiation-inducing factor.
[7] The production method of any of [1]-[6], wherein the TGFβ family signal transduction pathway inhibiting substance is a Nodal/Activin signal transduction pathway inhibiting substance, a TGFβ signal transduction pathway inhibiting substance, or a BMP signal transduction pathway inhibiting substance.
[8] The production method of any of [1]-[7], wherein the TGFβ family signal transduction pathway inhibiting substance is Lefty, SB431542, A-83-01 or LDN193189.
[9] The production method of any of [1]-[8], wherein the Sonic hedgehog signal transduction pathway activating substance is Shh, SAG or Purmorphamine.
[10] The production method of any of [1]-[9], wherein the differentiation-inducing factor in the third step is a BMP signal transduction pathway activating substance.
[11] The production method of [10], wherein the BMP signal transduction pathway activating substance is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.
[12] The production method of [10], wherein the BMP signal transduction pathway activating substance is BMP4.
[13] The production method of any of [1]-[12], wherein the aggregate obtained in the third step comprises a retinal tissue.
[14] The production method of any of [1]-[13], wherein the aggregate obtained in the third step comprises one or more cells selected from the group consisting of retinal progenitor cell, neural retinal progenitor cell, photoreceptor precursor cell, photoreceptor cell, rod photoreceptor cell, cone photoreceptor cell, horizontal cell, amacrine cell, interneuron, ganglion cell, retinal pigment epithelial cell, and ciliary marginal zone cell.
[15] The production method of [13] or [14], wherein the differentiation-inducing factor in the third step is a BMP signal transduction pathway activating substance, and the medium in the second step contains a Sonic hedgehog signal transduction pathway activating substance.
[16] The production method of [15], wherein the pluripotent stem cells are human pluripotent stem cells, the Sonic hedgehog signal transduction pathway activating substance in the medium in the second step has a concentration corresponding to Sonic hedgehog signal transduction activity of SAG at 10 nM-700 nM, and the retinal tissue is a human retinal tissue.
[17] The production method of [15] or [16], wherein, in the third step, the BMP signal transduction pathway activating substance is added to the medium between day 1 and day 9 after the start of the second step.
[18] The production method of [13] or [14], wherein, in the third step, the aggregate is cultured in a medium containing a Sonic hedgehog signal transduction pathway activating substance at a concentration not more than a concentration corresponding to Sonic hedgehog signal transduction activity of SAG at 700 nM.
[19] The production method of any of [1]-[9], wherein the differentiation-inducing factor in the third step is a TGFβ family signal transduction pathway inhibiting substance and/or a Wnt signal transduction pathway inhibiting substance.
[20] The production method of [19], wherein the TGFβ family signal transduction pathway inhibiting substance is Lefty, SB431542, A-83-01 or LDN193189.
[21] The production method of [19] or [20], wherein the Wnt signal transduction pathway inhibiting substance is IWR-1-endo.
[22] The production method of any of [1]-[9] or [19]-[21], wherein the aggregate obtained in the third step contains a cerebral tissue.
[23] The production method of any of [1]-[22], wherein a period for the culturing in the first step is 0.5 hr-144 hr.
[24] The production method of any of [1]-[23], wherein the first step is performed by an adhesion culturing method.
[25] The production method of any of [1]-[24], wherein, in the third step, the differentiation-inducing factor is added to the medium between day 3 and day 6 after the start of the second step.
[26] The production method of any of [1]-[15] and [17]-[25], wherein the pluripotent stem cells are primate pluripotent stem cells.
[27] The production method of any of [1]-[26], wherein the pluripotent stem cells are human pluripotent stem cells.
[28] The production method of any of [1]-[27], wherein the pluripotent stem cells are induced pluripotent stem cells.
[29] The production method of any of [1]-[28], wherein uniformed aggregates are formed in the second step.
[30] The production method of any of [1]-[29], wherein the suspension culturing is performed in the absence of a basement membrane preparation.
[31] A reagent for evaluating toxicity or efficacy of a test substance, comprising neural cells or a neural tissue produced by the method of any of [1]-[30].
[32] A method for evaluating toxicity or efficacy of a test substance, comprising bringing the substance into contact with neural cells or a neural tissue produced by the method of any of [1]-[30], and detecting an influence of the substance on the cells or tissue.
[33] A medicament for treating a disease due to a disorder of a neural cell or a neural tissue, comprising neural cells or a neural tissue produced by the method of any of [1]-[30].
[34] The medicament of [33], wherein the neural cells or neural tissue is retinal progenitor cells, retinal layer-specific neurons, retinal tissue, cerebral neural precursor cells, cerebral layer-specific neurons, or cerebral tissue.
[35] A method of treating a disease due to a disorder of a neural cell or a neural tissue, comprising transplanting an effective amount of neural cells or a neural tissue produced by the method of any of [1]-[30] to a subject in need of the transplantation.
[36] Neural cells or a neural tissue produced by the method of any of [1]-[30] for use in the treatment of a disease due to a disorder of a neural cell or a neural tissue.
[37] A pharmaceutical composition comprising neural cells or a neural tissue produced by the method of any of [1]-[30] as an active ingredient.

Effect of the Invention

According to the present invention, a high quality cell aggregate, as well as neural tissues such as retinal tissue and the like, and neural cells can be produced with high efficiency from pluripotent stem cells cultured in the absence of feeder cells.

