JP2014023457A - Nerve cell sheet and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nerve cell sheet exerting the function of nerve cells and a method for producing the same.SOLUTION: A nerve cell sheet has nerve cell layers formed by cultured nerve cells with extended axons. A method for producing the nerve cell sheet with nerve cell layers formed by nerve cells with extended axons includes induction of nerve cells from precursor cells by culturing the precursor cells in a culture solution containing a sonic hedgehog inhibitor and a differentiation inducer, and obtainment of nerve cell layers formed by cultured nerve cells with extended axons by culturing the nerve cells in a culture solution containing at least one of stromal cells-derived factor-1 (SDF1) and monocyte chemoattractant protein-1 (MCP-1) to induce axonal extension of the nerve cells.

Description

  The present invention relates to a nerve cell sheet and a method for producing the same.

Currently, the number of stroke patients is said to be about 1.5 million in Japan, and more than 250,000 people are newly diagnosed with stroke every year. In cerebrovascular diseases represented by stroke, for example, nerve cells (neurons) are damaged or killed in the cerebral cortex, so that it is considered very difficult to restore the function at the damaged site.
In general, as one of the methods for improving treatment or condition when a part of living tissue is damaged, it has been proposed to transplant cells or damaged tissue to the damaged site. Has been implemented.
As a material used for transplantation, for example, Patent Document 1 discloses a regenerated corneal endothelial cell sheet composed of corneal endothelial cells. Patent Document 2 discloses an epidermal cultured cell sheet composed of epidermal cells.
As for damage in the cerebral cortex, a method of injecting nerve cells into the damaged site can be mentioned (for example, see Non-Patent Document 1).

  In addition, as a treatment for neurological diseases, medical treatment for regenerating damaged central nerves by transplanting neural stem cells and / or neural progenitor cells has attracted attention. For this treatment, neural stem / progenitor cell proliferation promoters (for example, see Patent Document 3) and differentiation promoters (for example, see Patent Document 4) have been developed and their use has been proposed.

International Publication No. 2004/073761 Pamphlet International Publication No. 2002/010349 Pamphlet JP 2006-254753 A JP 2012-75380 A

Inflammation and Regeneration, 2010, Vol.30, No.3, pp.193-205

  However, unlike epidermis cells and the like, a complex structure such as axon, myelin sheath, and synapse formation is necessary for a neuron to function functionally. It is also known that nerve cells having such a complex structure are special cells that exhibit their functions for the first time when they form a network and interact with each other. For this reason, in order to use a nerve cell as a transplant, even if it is injected into a damaged site in a single cell state, the expected effect may not be obtained.

  Accordingly, an object of the present invention is to provide a nerve cell sheet in which the function of nerve cells is effectively exhibited and a method for producing the same.

The present invention is as follows.
[1] A nerve cell sheet having a nerve cell layer formed by cultured nerve cells in which axons are extended.
[2] In the above [1], the cultured neuron cell is a cell derived from at least one progenitor cell selected from the group consisting of an embryonic stem cell (ES cell) and an induced pluripotent stem cell (iPS cell). The nerve cell sheet described.
[3] Progenitor cells are cultured in a culture medium containing a sonic hedgehog inhibitor and a differentiation-inducing agent to induce neurons from the precursor cells, and the neurons are transformed into SDF1 (Stromal cells-derived factor). -1) and MCP-1 (Monocyte chemoattractant propein-1) cultured in a culture medium containing at least one, induces axonal extension of the nerve cells, and nerves formed by cultured neurons that have axonal extension Obtaining a cell layer, The method for producing a nerve cell sheet according to [1] or [2].
[4] The method for producing a nerve cell sheet according to [3], wherein the concentration of at least one of the SDF1 and MCP-1 in the culture solution is 0.5 ng / mL to 30 ng / mL.
[5] The method for producing a nerve cell sheet according to [3] or [4], wherein the sonic hedgehog inhibitor includes at least one selected from the group consisting of cyclopamine, jervin and tomatidine.
[6] The production of the nerve cell sheet according to any one of [3] to [5], wherein the concentration of the sonic hedgehog inhibitor in the culture solution is 2.0 × 10 ⁻⁷ μM to 3.0 μM. Method.
[7] The differentiation inducer according to [3] to [6], wherein the differentiation inducer includes at least one selected from the group consisting of retinoic acid, noggin, fibroblast growth factor (FGF), and nerve cell growth factor (NGF). The manufacturing method of the nerve cell sheet in any one.
[8] The progenitor cells are at least one selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells and neural progenitor cells [3] to [7 ] The manufacturing method of the nerve cell sheet in any one of.
[9] A sheet material for transplantation comprising the nerve cell sheet according to [1] or [2].

  ADVANTAGE OF THE INVENTION According to this invention, the nerve cell sheet in which the function of a nerve cell is exhibited and its manufacturing method can be provided.

It is an optical microscope photograph image of the nerve cell sheet concerning Example 1 (20 times). It is an optical microscope photograph image of the nerve cell sheet concerning Example 1 (2 times). It is an optical microscope photograph image of the nerve cell sheet | seat from the mouse | mouth ES cell strain concerning Example 3 (20 time). It is a photograph image of the nerve cell sheet | seat from the mouse | mouth iPS cell concerning Example 3 (2 times). It is an optical microscope photograph image of the nerve cell sheet | seat from the mouse | mouth embryonic neural progenitor cell concerning Example 3 (20 time).

The nerve cell sheet of the present invention is a nerve cell sheet having a nerve cell layer formed by cultured nerve cells in which axons are extended.
Moreover, the method for producing a nerve cell sheet of the present invention comprises culturing progenitor cells in a culture solution containing a sonic hedgehog inhibitor and a differentiation inducer, inducing nerve cells from the progenitor cells, and Culturing the nerve cell in a culture medium containing at least one of SDF1 (Stromal cells-derived factor-1) and MCP-1 (Monocyte chemoattractant propein-1) to induce axonal extension of the nerve cell; A method for producing a nerve cell sheet having a nerve cell layer formed by a nerve cell extended by axon, comprising obtaining a nerve cell layer formed by cultured nerve cells extended by axon.

  The nerve cell sheet is not a collection of individual nerve cells, but has a nerve cell layer formed by cultured nerve cells in which axons have been extended. Is retained. For this reason, unlike the case of using a nerve cell in a state where the cell-cell interaction is not maintained, it is expected that the nerve cell exhibits a high function.

  To explain this further, when the nerve cell finally differentiates after proliferation, it becomes a form having a protrusion capable of axon extension and stops proliferation. In order for nerve cells to exert their functions, it is indispensable to transmit information between nerve cells, and an axon extended form is essential. Since the nerve cell sheet according to the present invention has a nerve cell layer formed by such cultured axon-extended nerve cells, the nerve cell-cell interaction is retained and the nerves collected using trypsin are retained. Compared with the conventional method in which cells are directly administered, the engraftment rate of nerve cells is increased, and a high function based on the cell-cell interaction of neurons is exhibited.

  In addition, the method for producing a nerve cell sheet of the present invention induces a nerve cell from a precursor cell in a culture solution containing a sonic hedgehog inhibitor and a differentiation inducer, and also includes a culture containing at least one of SDF1 and MCP-1. Inducing axonal extension of nerve cells in the liquid to obtain a layer formed by cultured nerve cells that have extended axons, so that it has a nerve cell layer that contains a high proportion of cultured neurons that have axonal extension, As a result, a nerve cell sheet capable of expressing the function of nerve cells can be efficiently produced.

In the present invention, with respect to the nerve cell layer, “formed by nerve cells” means that the axons of the plurality of nerve cells extended by culturing a plurality of nerve cells that have undergone final differentiation under predetermined conditions are mutually connected. By connecting, it means that the nerve cell layer is mainly composed.
In the present invention, the term “axon extended” means a state in which nerve cells are integrated with each other by constructing a shaftwork in a mesh shape.

In this specification, the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
In the present specification, “to” indicates a range including the numerical values described before and after the values as a minimum value and a maximum value, respectively.
Furthermore, in this specification, the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. means.
The present invention will be described below.

[Neuron cell sheet]
The nerve cell sheet according to the present invention has a nerve cell layer formed by cultured nerve cells in which axons are extended.

<Neuron cell layer>
The nerve cell layer is formed by cultured nerve cells in which axons are extended.
A nerve cell (neuron) is a cell having a complex structure composed of a cell body and a process (dendrites and axons), and from a functional aspect, it is classified into three types of motor neurons, intervening neurons, and sensory neurons. Divided. The nerve cells forming the nerve cell layer may be any of motor neurons, intervening neurons and sensory neurons, or a combination thereof. From the viewpoint of repair of motor function, a motor nerve cell is preferable. Examples of motor neurons include cortical motor neurons and spinal motor nerves, and cortical motor neurons are more preferable from the viewpoint of therapeutic effects on cerebral cortical dysfunction.

  The nerve cell layer may include nerve cells that are not axonally extended within a range in which the shape of the layer can be maintained. It can be confirmed by the optical microscope and the actual microscope that the nerve cells are axon-extended based on the form. From the viewpoint of effective functional expression of the nerve cell sheet, the content of nerve cells that are axon-extending is preferably 70% or more of the total number of neurons contained in the nerve cell layer, More preferably, it is 80% or more. The ratio of the number of nerve cells having axonal extension in the total number of nerve cells can be confirmed by staining with hematoxylin and eosin.

The neural cell is a cell derived from at least one progenitor cell selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells and neural progenitor cells. Is preferable from the viewpoint of the amount of cells supplied.
The progenitor cells may be any cells that can be induced into nerve cells, such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), fetal nerves Examples thereof include cells, neural stem cells, neural progenitor cells and the like, and these can be used alone or in combination. Among these, ES cells and iPS cells are preferable in terms of the amount of cells supplied.

  These cells are used in various animals such as mammalian primates (eg, humans, monkeys, etc.), ungulates (eg, pigs, cows, horses, etc.), small mammal rodents (eg, mice, rats, rabbits, etc.). Can be appropriately selected according to the purpose of use. The cell supply source is not limited to adults, but may be derived from a fetus.

  Embryonic stem cells refer to pluripotent stem cells derived from early embryos, and are characterized by infinite proliferation ability and differentiation ability into multiple cell lines. Embryonic stem cells can be isolated from a fertilized egg or an early embryo at a stage from the fertilized egg to a blastocyst that has developed, and can be obtained from, for example, RIKEN.

  iPS cells are pluripotent stem cells prepared by introducing genes such as OCT3 / 4, SOX2, KLF4, and C-MYC into somatic cells (Cell, Vol. 131: pp. 861-872). Here, “pluripotency” means the ability to differentiate into a plurality of cell lines, and can be confirmed by a teratoma formation method. A commercially available iPS cell line can be used, and for example, it can be obtained as a 253G1 strain (human), 201B7 strain (human), etc. from RIKEN BioResource Center. Further, the iPS cell line may be an iPS cell newly prepared from human patient skin fibroblasts according to the above-mentioned document or the like. iPS cells and cells derived from iPS cells can be confirmed by detecting OCT3 / 4, SOX2, KLF4, C-MYC, and the like.

  Neural stem cells are stem cells that have the ability to supply cells that differentiate into neurons and glial cells. Neural progenitor cells are undifferentiated cells that differentiate only into nerves. Since these cells are difficult to distinguish strictly, they are applied without distinction in the present invention. Any of the neural stem cells or neural progenitor cells can be obtained by, for example, inducing differentiation from human iPS cells. Neural stem cells or cells derived from neural stem cells, or neural progenitor cells or cells derived from neural progenitor cells can be confirmed by nestin protein expression and NCAM positivity using a flow cytometer.

  Being a neuron can be confirmed by detecting a molecule known as a neuron marker in addition to the confirmation based on the characteristic form described above. For example, CTIP2 (COUP (Chicken ovalbumin upstrem promoter) -TF (transcription factor) -interaction protein 2), Fezf2 (FEZ (Forebraiin embryonic zinc finger) family zing finger 2), FoxP2 (forkhead box) P2) and Crim1 (Cystein-rich motor neuron 1 protein precursor). The presence of these markers can be confirmed by known methods such as gene expression or protein detection.

  The nerve cell layer is sufficient if at least 50% or more of the cells constituting the nerve cell layer are the nerve cells, and can include cells other than nerve cells as long as the shape of the layer (sheet) can be maintained. In terms of the strength and functionality of the nerve cell sheet, the ratio of the nerve cells in the total cells constituting the nerve cell sheet is preferably 70% or more, more preferably 80% or more, 85% More preferably, it is more preferably 95% or more. Increasing the proportion of neurons in all the cells that make up the sheet tends to reduce the frequency of mixing of other types of cells, improving the strength of the sheet, and increasing the function expression efficiency per unit area. is there.

Other types of cells that can be contained in the nerve cell layer include vascular endothelial cells, fibroblasts, oral mucosal epithelial cells, tracheobronchial epithelial cells, gastric mucosal epithelial cells, corneal epithelial cells, intestinal mucosal epithelial cells, etc. Examples include various epithelial cells. When these cells are included, it is preferable to perform known growth suppression treatment such as radiation irradiation on cells other than nerve cells.
Wherein the neurological The number of cells cell layer constituting not particularly limited, for example, be a 0.1 or ^/ cm 2 ^~0.1 × 10 ⁶ cells / cm ^2.
The nerve cell sheet is preferably composed of only a nerve cell layer in consideration of transplantation and the like, but may include other layers other than the nerve cell layer. Examples of other layers include cell layers including fibroblasts or epithelial cells. By including the layer of these other cells, the state of the nerve cell contained in the nerve cell layer can be improved by factors from other cells.

There is no restriction | limiting in particular about the thickness and magnitude | size of the said neuronal cell sheet | seat, It can prepare suitably as needed. The thickness of the nerve cell layer in the nerve cell sheet can generally be in the range of 0.05 mm to 1 mm.
The size and thickness of the nerve cell layer of the nerve cell sheet can adjust the thickness of the nerve cell layer by adjusting the culture period in the manufacturing method described later, the cell seeding density, and the like. . The thickness of the nerve cell layer can be confirmed under a microscope, for example, by preparing a section of a nerve cell sheet. In the case of having a layer of other cells, it can be appropriately adjusted by adjusting the culture period, the cell seeding density, etc., similarly to the thickness and size of the nerve cell layer.

[Manufacturing method of nerve cell sheet]
In the method for producing a nerve cell sheet according to the present invention, precursor cells are cultured in a culture solution containing a sonic hedgehog inhibitor and a differentiation inducer, and nerve cells are induced from the precursor cells (hereinafter referred to as cells). And may be referred to as an induction step), and the neurons are cultured in a culture medium containing at least one of SDF1 (Stromal cells-derived factor-1) and MCP-1 (Monocyte chemoattractant propein-1), Inducing axonal extension of nerve cells and obtaining a nerve cell layer formed by cultured neurons that have axonal extensions (hereinafter sometimes referred to as axonal extension induction process) A method for producing a nerve cell sheet having a nerve cell layer formed by cells.

  In the cell induction step, nerve cells are induced from progenitor cells in a culture solution containing a sonic hedgehog inhibitor and a differentiation inducer. Sonic hedgehog is known as a protein used for differentiation induction. However, in the production method described above, a nerve cell is frequently generated from progenitor cells by combining a sonic hedgehog inhibitor and a differentiation inducer. Can be guided to.

  Examples of the sonic hedgehog inhibitor include cyclopamine, gerbin, tomatidine, and the like, and these can be used alone or in combination of two or more. Among these sonic hedgehog inhibitors, cyclopamine and jervin are preferable, and cyclopamine is more preferable in terms of induction efficiency of cortical motor neurons.

The concentration of the sonic hedgehog inhibitor in the culture solution varies depending on the type of the sonic hedgehog inhibitor used. In the case of cyclopamine, the concentration is 2.0 × 10 ⁻⁷ μM to 3.0 μM. It is preferable that it is 2.5 * 10 < ^-5 > micromol-2.5 * 10 < ^-2 > micromol. If the concentration is 2.0 × 10 ⁻⁷ μM or more, there is a tendency that nerve cells can be reliably induced from the progenitor cells. If the concentration is 3.0 μM or less, cell death is more reliably suppressed and sufficient. There is a tendency to induce a large number of neurons. In particular, by setting it to 2.5 × 10 ⁻² μM or less, it can be surely induced into motor neurons.

  As the differentiation inducer, any differentiation inducer known to contribute to differentiation induction from progenitor cells to nerve cells can be used. Examples of such differentiation inducers include retinoic acid (RA), noggin, fibroblast growth factor (FGF), and nerve cell growth factor (NGF). These may be used alone or in combination of two. The above can be used in combination. From the viewpoint of the induction efficiency to nerve cells, it is preferable to use a combination of two or more selected from the group consisting of retinoic acid, noggin and FGF, and more preferably a combination of retinoic acid and noggin. Further, noggin, FGF and NGF may be natural products or gene recombinants. These differentiation-inducing agents may be added as the compound itself, or may be those chemically modified from the viewpoint of stability and reactivity or those in a multimeric form. For example, in the case of Noggin, it may be used as a trimer, or may be a conjugate with a constant region (Fc) of an immunoglobulin heavy chain.

  The concentration of the differentiation inducer in the culture solution may be any concentration that can induce differentiation from progenitor cells to neurons, and a known concentration can be used as it is for each differentiation inducer. For example, in the case of retinoic acid, it is preferably 0.01 μM to 10 μM, more preferably 1 μM to 5 μM. In the case of Noggin, it is preferably 1 ng / ml to 1,000 ng / ml, more preferably 100 ng / ml to 500 ng / ml.

  The progenitor cells may be any cells that can be induced into nerve cells, such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells. , Neural progenitor cells and the like, and these can be used alone or in combination. Among these, ES cells and iPS cells are preferable in terms of the amount of cells supplied. For progenitor cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells, and neural progenitor cells that can be used as the progenitor cells, The matters described can be applied as they are.

The culture solution used in the differentiation induction step can be appropriately selected depending on the type of cells to be cultured. The culture medium that can be used may be any known one, and examples thereof include DMEM, MEM, F12, and DME medium. In addition, various components that can be generally added, such as glucose, serum components, antibiotics (penicillin, streptomycin, etc.), etc., may be added to these culture solutions. In addition, although the density | concentration in the case of adding serum can be suitably changed according to the culture state at that time, it can usually be 10% (v / v). When culturing ES cells or iPS cells, it is preferable not to contain serum, and it is preferable to use a knockout serum substitute (KSR).
For cell culture, normal culture conditions, for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% CO ₂ are applied.

The incubator used for the culture in the differentiation induction step is not particularly limited as long as it is a normal incubator for culturing the type of cells used. For example, in the case of embryonic stem cells or iPS cells, from the viewpoint of growth efficiency, an incubator equipped with an adherent culture surface is preferable.
As an incubator having such an adherent culture surface, for example, a culture surface coated with a cell adhesion factor such as fibronectin (FN), vitronectin (VN), laminin, or a water-soluble polymer compound such as gelatin is used. Examples include incubators having the same.

As the seeding density of the progenitor cells in the incubator, from the viewpoint of proliferation efficiency, for example, in the case of a 10 cm petri dish, 0.5 × 10 ⁵ / petri dish to 1.0 × 10 ⁷ pieces / petri dish can do.

  Depending on the type of the progenitor cells, the cells may be cultured with feeder cells from the viewpoint of proliferation efficiency and the like. Any feeder cells can be used as long as they do not impair the proliferation of progenitor cells and the function of the nerve cell sheet, and generally include mouse embryos, fibroblasts and the like. In addition, the feeder cells are preferably those that have been subjected to growth inhibition treatment by irradiation and use of a drug such as mitomycin C.

  The culture period for differentiation induction can be 8 to 30 days, for example. Induction of differentiation into nerve cells can be confirmed by the expression of nestin, neurofilament M chain, and βIII tubulin by PCR and immunostaining.

  Prior to the differentiation induction step, a preculture step may be provided for the purpose of satisfying a predetermined number of cells. As the preculture step, known culture conditions that can proliferate progenitor cells before differentiation induction can be applied. The preculture conditions vary depending on the type of target cells, but those skilled in the art can appropriately select from known conditions such as medium composition, culture temperature, and culture period.

The axon extension inducing step includes culturing the nerve cell in a culture solution containing at least one of SDF1 and MCP-1 to obtain a nerve cell layer formed by the axon-extended nerve cell. Since the nerve cells are cultured in a culture medium in which SDF1 and / or MCP-1 are present, nerve cell axon extension occurs efficiently.
The axon extension induction step is a step performed to extend (elongate) the axons of the nerve cells that have undergone differentiation in the differentiation induction step. In a culture that does not have the axon extension induction step, the nerve cell sheet is used. Can't get.

  The axonal extension inducing step may be performed after the differentiation from progenitor cells to neurons has been sufficiently completed in the differentiation inducing step, or may be performed simultaneously with the differentiation inducing step. When the axonal extension induction step is performed simultaneously with the differentiation induction step, a differentiation inducer used in the differentiation induction step may be included in the culture solution used in the axonal extension induction step.

SDF1 (Stromal cell-derived factor 1) is one type of cytokine that performs cell proliferation, differentiation and functional expression (Science, 1993, vol.261, pp.600-603). Contacting SDF1 with nerve cells causes maturation of nerve cells and axons extend.
MCP-1 (Monocyte chemoattractant protein-1: Monocyte chemoattractant protein-1) is a kind of chemokine consisting of 92 to 99 amino acids having basic and high heparin binding properties (Proc. Natl. Acad. Sci. USA, Vol.91, pp.3652-3656, 1994). Contacting MCP-1 with nerve cells causes maturation of nerve cells and axons extend.

  SDF1 and MCP-1 can be used alone or in combination of two or more. From the viewpoint of the axon extension efficiency and survival rate corresponding to the type of cells used, it is preferable to use them alone or in combination, and it is more preferable to use only SDF1.

  The concentration of SDF1 and / or MCP-1 in the culture solution in the axon extension induction step is preferably 0.5 ng / mL or more in total in terms of axon extension efficiency and survival rate, More preferably, it is 1.0 ng / mL to 10 ng / mL. If it is 0.5 ng / mL or more, axonal extension can be reliably induced. Although there is no restriction | limiting in particular about an upper limit, It is preferable to set it as 30 ng / mL or less at the point of the efficient effect expression with respect to addition amount.

  In particular, when SDF1 is used alone, the concentration in the culture solution is preferably 0.5 ng / mL to 30 ng / mL from the viewpoints of axonal extension efficiency and survival rate. Moreover, it is preferable that the density | concentration in the culture solution when using MCP-1 independently is 0.5 ng / mL-30 ng / mL from a viewpoint of axon extension efficiency and a survival rate.

  As the culture conditions in the axonal extension induction process, those mentioned as the culture conditions in the differentiation induction process can be used as they are. Moreover, as a culture solution which can be used in an axonal extension induction | guidance | derivation process, the culture solution and additive component which were described in the differentiation induction | guidance | derivation process can be applied as it is, except not including a sonic hedgehog inhibitor and a differentiation inducer.

The period of the axon extension guidance step is not particularly limited as long as it is a period in which axon extension can be confirmed, but may be, for example, 8 to 30 days.
Here, the timing of addition of SDF1 and / or MCP-1 to the medium can be added every day after the start of culture, and more preferably every 3 days.
The axonal extension of the nerve cell can be confirmed with an optical microscope and an actual microscope as described above.

  For the culture in the axonal extension induction step, it is preferable to use an incubator equipped with a release layer on the culture surface from the viewpoint of cell sheet production efficiency. When using an incubator equipped with a release layer on the culture surface, a cell sheet is formed on the surface of the release layer by seeding cells on the release layer and culturing. Thereafter, the culture surface and the cell sheet can be separated from each other based on the function of the release layer, and the cell sheet can be easily detached from the incubator.

Examples of the release layer that can separate the culture surface and the cell sheet include those capable of decomposing or disappearing, for example, collagen gel, temperature-reactive polymer, and the like. . From the viewpoint of simplicity and shape maintenance, a release layer made of a temperature-reactive polymer is preferable.
Examples of such temperature-responsive polymers include hydrogels such as poly-N-isopropylacrylamide (PIPAA) or derivatives thereof. For example, since PIPAA becomes liquefied when the temperature is 32 ° C. or lower, for example, the release layer can be liquefied by low-temperature treatment at 20 ° C. for 60 minutes to release the nerve cell sheet from the culture surface. Examples of such a temperature-responsive polymer include meviol gel (registered trademark) and BST-Gel (registered trademark). After dissolving in ultrapure water, it is adsorbed on plastic.
Moreover, the incubator which has a multilayer peeling layer which combined such a temperature-responsive polymer, other collagen gels, etc. may be sufficient. When collagen gel or the like is used, the cell sheet can be peeled by decomposing the gel by using an enzyme or the like.

After the axon extension inducing step, a nerve cell sheet having a nerve cell layer formed from nerve cells that have axon extended is obtained. The method for producing a nerve cell sheet may include separating the nerve cell sheet from the culture surface of the incubator and collecting it (hereinafter referred to as a collecting step).
The recovery of the nerve cell sheet in the recovery step may be performed by any means as long as the nerve cell sheet can be separated from the culture surface. For example, the sheet may be separated by a method such as picking up the end of the sheet, or gently peeled by gentle pipetting or the like. When an incubator having a release layer is used as the incubator, the nerve cell sheet can be separated by dissolving or decomposing the release layer.

As another method for producing the nerve cell sheet, there is a method of producing a nerve cell after final differentiation by culturing it in a predetermined incubator. In this case, if necessary, an inducer that induces axonal extension may be added to the culture solution.
About the culture solution which can be used with this other manufacturing method, what was mentioned above is applicable as it is. Examples of the inducer that induces axonal extension include the above-described SDF1, MCP-1, and the like. When the inducer is used for nerve cells that have undergone final differentiation, the above-described concentrations may be applied as they are.

  The nerve cell sheet obtained by the above production method has a nerve cell layer formed by nerve cells that are axon-extended and that retain cell-cell interactions. The high functionality of such a nerve cell sheet can be confirmed by the high content of nerve cells in the nerve cell sheet, the recovery evaluation of nerve activity when transplanted as a graft, and the like. As the recovery evaluation of the nerve activity, a test well-known in the art may be applied. The evaluation test varies depending on the type of nerve cells constituting the nerve cell sheet, but in the case of motor neurons, a beam walking test, a rotating rod test, or the like can be given.

[Usage]
The nerve cell sheet retains the cell-cell interaction of nerve cells, has a higher engraftment rate than the conventional method directly using cells isolated using trypsin, and easily exerts the function of nerve cells, High functionality. For this reason, the said nerve cell sheet is preferably used for the use according to the function of the nerve cell.
For example, treatment of diseases or conditions associated with nerve cell damage. Examples of such diseases include cerebral hemorrhage, stroke, cerebral infarction, cerebral contusion and the like. In the treatment of such a disease or symptom, it is expected that the nerve cell sheet is transplanted to the site to be treated to exert a therapeutic effect at the site to be treated. When the nerve cell sheet is transplanted, the nerve cells constituting the nerve cell layer of the nerve cell sheet communicate with the nerve cells of the treatment target site by cell-cell interaction, or the nerve cells infiltrate the treatment target site, It is speculated that a therapeutic effect is obtained, but is not bound by this theory.

The present invention also includes a method for treating a disease or symptom associated with nerve cell damage using the nerve cell sheet.
The treatment method includes transplanting the nerve cell sheet in a local area where nerve cell damage has occurred or may have occurred. As the transplantation method, the nerve cell sheet may be transplanted by any method as long as it can be applied to the damaged site.
According to the treatment method, since the nerve cell sheet in which the cell-cell interaction is maintained is directly applied to the damaged site, the function based on the nerve cell interaction is rapidly exerted, and the disease caused by the nerve cell damage is prevented. The condition or symptom is effectively improved.

  In addition, the nerve cell sheet can be used as a sheet material for transplantation that can be applied to an affected area in the form as it is. That is, the transplant sheet material according to the present invention includes the nerve cell sheet. By using the present sheet material for transplantation, engraftment of nerve cells is good and a high therapeutic effect can be expected.

  The nerve cell sheet preferably includes layers other than the nerve cell layer from the viewpoint of handling before transplantation, for example. Examples of such other layers include a base material layer in order to improve the strength of the sheet. Examples of the substrate used for the substrate layer include a membrane such as a nitrocellulose membrane. There is no restriction | limiting in particular as the thickness of the said base material layer, For example, if it is a layer of a feeder cell, it can be 0.05 m-1 mm.

When the nerve cell sheet has a layer other than the nerve cell layer, the method for producing a nerve cell sheet of the present invention can further include a step of adding the other layer to the nerve cell layer. .
For example, when the nerve cell sheet has a base material layer, a step of providing a base material on the nerve cell layer obtained by the axonal extension induction can be included. The application of the base material varies depending on the type of base material used, but for example, when a membrane is used as the base material, it can be placed on the nerve cell layer. In addition, after placing the membrane on the culture surface in advance, the nerve cells may be seeded, and then the axonal extension inducing step may be performed.
Thereby, a nerve cell sheet having another layer can be obtained.

  The nerve cell sheet can also be used for determination of the effect of drugs on nerve cells and academic research. For example, in the method for determining the effect of a drug on nerve cells, the candidate substance is brought into contact with the nerve cell sheet, the nerve cell sheet after contact is subjected to a predetermined physiological activity test, and the candidate substance is based on the result of the test. Determining that is a target substance. This makes it possible to easily and well determine whether or not the compound has a predetermined physiological activity on nerve cells.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them.

[Example 1]
(1) Preparation of progenitor cells (1-1) Maintenance of human iPS cells 1. Human iPS cell line 253G1 or 201B7 (obtained from RIKEN) was placed on feeder cells (mouse fetal fibroblasts treated with mitomycin C). DMEM / F12 (GIBCO) seeded at a cell density of 0 × 10 ⁶ cells / 10 mL, supplemented with 20% (v / v) knockout serum (KSR: Life Technologies Japan), non-essential amino acid mixture, pyruvic acid The product was maintained at 37 ° C. and 5% (v / v) CO ₂ for maintenance (maintenance culture). The medium was changed every day.
Among the iPS cells maintained as described above, those having the number of passages within the 50th generation and having a normal karyotype were used for the following nerve cell induction.

(1-2) Monkey ES cells A monkey ES cell line (obtained from Kyoto University) was subjected to maintenance culture in the same manner as in (1-1) above, and used for the following nerve cell induction.

(2) Differentiation induction method from progenitor cells to nerve cells and preparation of sheet The above-mentioned human iPS cells and monkey ES cells were collected and seeded on a bacterial grade plastic petri dish (Falcon) for maintenance. Using the same medium as the culture, the culture in a floating state was started. After 4 days, floating cell aggregates (EB), which are spherical cell clusters, were obtained. This was recovered and transferred to a fresh fibronectin-coated Petri dish (Becton Dickinson) together with the above medium. After confirming cell attachment, the final concentration of 1 μM retinoic acid (RA: Sigma) and a final concentration of 7 ng / mL Noggin-Fc (manufactured by R & D) and cyclopamine (Sigma Aldrich) having a final concentration of 2.5 × 10 ⁻² μM were added (Day 1). Thereafter, on the third day, the medium containing RA, Noggin-Fc and cyclopamine was added to the Petri dish so as to have the same final concentration as above.

On the fourth day of culture, the cells obtained above were placed in a 48-well culture plate (Meviol Co.) previously coated with a temperature-responsive hydrogel (Meviol Gel (registered trademark), Meviol Co., Ltd.) with 1 × Seed and cultured at a cell density of 10 ⁵ / well. Every 3 days, a medium containing SDF1 was added so that the final concentration of SDF1 (Abcam) was 1 ng / ml. After culturing for about 14 to 21 days, the temperature is changed to 20 ° C. to dissolve the hydrogel, and lightly pipetting is performed, and a nerve cell sheet is formed from the culture surface of each well using a nitrocellulose membrane. The iPS cell-derived nerve cell sheet and the ES cell-derived nerve cell sheet were each collected.

The properties of the obtained nerve cell sheet were confirmed as follows.
That is, the nerve cell sheet obtained above was collected. Using trypsin, the cells were stained with a PE-labeled anti-human NCAM antibody (Becton Dickinson) as a floating cell population, and the number of NCAM-expressing cells was confirmed by flow cytometry.

  Furthermore, against the obtained nerve cell sheet, an antibody (ABCAM) against CTIP2 (COUP (Chicken ovalbumin upstrem promoter)-TF (transcription factor)-interaction protein 2), Fezf2 (FEZ (Forebraiin embryonic zinc finger) family zing Staining was performed with an antibody against finger 2) (Santa Cruz), an antibody against FoxP2 (forkhead box P2) (Santa Cruz), and an antibody against Crim1 (Cystein-rich motor neuron 1 protein precursor) (Santa Cruz).

The nerve cell sheet obtained using 2.5 × 10 ⁻⁵ μM cyclopamine was observed with an optical microscope (Carl Zeiss). The results are shown in FIG. 1 (20 times) and FIG. 2 (2 times). As shown in FIG. 1, it can be seen that the axons of individual nerve cells can be confirmed and are in contact with other nerve cell axons to form a network.
Further, in the nerve cell sheet, 60% or more of the total number of cells expresses NCAM, which indicates that the nerve cell sheet contains nerve cells at a high content rate. Furthermore, it was confirmed by the staining of each antibody described above that the nerve cells constituting the nerve cell sheet were human motor neurons. In addition, the feeder cell was less than 0.01% by visual observation by fluorescent immunostaining (Confocal Scanning Microscope). As described above, it was confirmed that the entire nerve cell sheet was composed of a nerve cell layer formed by nerve cells that had axonal extension.

(3) Experimental brain injury due to cold injury and transplantation Female C57BL / 6 mice (6-8 weeks old) were used for transplantation. All subsequent procedures were performed in accordance with St. Marianna University School of Medicine guidelines.
Mice were anesthetized and placed in a stereotaxic frame (Narishige, Tokyo, Japan). The cephalic site was the left parietal bone between the outer 3.0 mm of corona, lambda and sagittal sutures.
A metal probe cooled with liquid nitrogen was applied to the surface of the burr site as it was, with a force of 100 grams four times for 30 seconds. By administering cold injury to each mouse four times, hemiplegia could be reliably reproduced, and a cerebral infarction model mouse was produced.

The iPS cell-derived nerve sheet or monkey ES cell-derived nerve cell sheet obtained above was transplanted into the left cerebral motor area of this cerebral infarction model mouse, respectively.
After adjusting the size of the nerve sheet to 1 mm to 2 mm square, the nerve sheet was spread on the damaged motor cortex and allowed to stand. Thereafter, it was confirmed that there was no bleeding. An artificial bone was placed thereon, or the scalp was sutured without placing the artificial bone, and the operation was completed.
For immunosuppression, cyclosporine (subcutaneous injection) was administered at a concentration of 10 μg / g and dexamethasone (intraperitoneal injection) was administered at a concentration of 0.2 μg / g for 1 hour before transplantation. . From the day after transplantation, cyclosporine was administered daily at a concentration of 10 μg / g.
Following transplantation, the following motor function evaluation was performed at a predetermined time.

(4) Motor function evaluation After transplantation, the motor function of the upper and lower limbs was evaluated by a beam walking test.
Specifically, the mouse after transplantation is placed on a wooden stick of 7 to 8 mm in width and 120 mm in length (set to a height of 300 mm on a 100 mm soft pad), and about 50 steps are taken on it. The number of times the upper and lower limbs failed to grab the wooden stick during that time was counted. The results are shown in Table 1.

In addition, the motor function and muscle coordination after transplantation were evaluated by a rotation test.
Specifically, a rotating unit (Muromachi) having a rotating rod having a diameter of 3.5 cm was used. The rotating rod was rotated at 30 rpm, the mouse was run on the rotating rod, and the time until falling was measured. The results are shown in Table 2.

  Similarly, the beam walking test and the rotation test were performed on mice in which 5 μl of a phosphate buffer (PBS) was injected in place of the nerve cell sheet at the same site as the nerve cell sheet transplanted in the transplanted mouse. went. The results are shown in Tables 1 and 2, respectively.

  As shown in Tables 1 and 2, recovery of motor function was observed in mice transplanted with nerve cell sheets.

[Example 2]
The iPS cell line used in Example 1 above was used, and the concentration of cyclopamine was 2.5 μM, 2.5 × 10 ⁻⁴ μM, 2.5 × 10 ⁻⁵ μM, or 2.5 × 10 ^−7. A neuronal cell sheet of the iPS cell line was prepared in the same manner as in Example 1 except that it was changed to μM.
As a result, an iPS cell-derived nerve cell sheet could be obtained regardless of the concentration of cyclopamine. In particular, when the concentration of cyclopamine is changed to 2.5 × 10 ⁻⁴ μM, 2.5 × 10 ⁻⁵ μM, or 2.5 × 10 ⁻⁷ μM, the axons are sufficiently extended. Therefore, there is a higher expectation for the high performance of nerve cells that have extended axons.

  In addition, the nerve cell sheet obtained by inducing differentiation with 2.5 μM cyclopamine has a higher contamination rate of vascular endothelial cells than other cell sheets, so that the cell sheet cannot be broken from the culture surface of the incubator. Care must be taken to peel off.

[Example 3]
Mouse ES cell line (Science. 1991 Nov 1; 254 (5032): 707-710, E14.1, Passage number 11-15, normal karyotype: provided by Dr. Klaus Rajewsky (Cologne University)), mouse iPS cells (RIKEN) Laboratory) and mouse fetal neural progenitor cells (Cosmo Bio Inc.), as in Example 1, a final concentration of 1 μM retinoic acid (RA: manufactured by Sigma) and a final concentration of 7 ng / mL Noggin-Fc ( R & D) and cyclopamine with a final concentration of 2.5 × 10 ⁻² μM were used to induce differentiation, and a nerve cell sheet was prepared using SDF1 with a final concentration of 1 ng / ml.

The culture conditions for the mouse ES cell line, mouse iPS cell, and mouse embryonic nerve cell are the same as those for the human iPS cell line of Example 1. The obtained nerve cell sheets are shown in FIGS. 3 is a nerve cell sheet from a mouse ES cell line (light microscope, 20 times), FIG. 4 is a nerve cell sheet from a mouse iPS cell (2 times), and FIG. 5 is a nerve cell from a mouse embryonic neural progenitor cell. A sheet (optical microscope, 20 times) is shown.
As shown in FIG. 3 to FIG. 5, a nerve cell sheet in which axons were extended from any of the progenitor cells could be obtained.

  As described above, the nerve cell sheet of the present invention is composed of a nerve cell sheet in which axons are extended, and by transplanting this nerve cell sheet, the function at the damaged site was remarkably recovered. From this, it can be seen that the nerve cell sheet of the present invention has high functionality.

Claims (9)

  A nerve cell sheet having a nerve cell layer formed by cultured nerve cells in which axons are extended.   2. The nerve cell according to claim 1, wherein the cultured nerve cell is a cell derived from at least one progenitor cell selected from the group consisting of an embryonic stem cell (ES cell) and an induced pluripotent stem cell (iPS cell). Sheet. Culturing progenitor cells in a culture medium containing a sonic hedgehog inhibitor and a differentiation inducer to induce neurons from the progenitor cells; and
Culturing the nerve cell in a culture medium containing at least one of SDF1 (Stromal cells-derived factor-1) and MCP-1 (Monocyte chemoattractant propein-1) to induce axonal extension of the nerve cell; Obtaining a nerve cell layer formed by cultured neurons that have axonal extensions;
The manufacturing method of the nerve cell sheet | seat of Claim 1 or Claim 2 containing this.   The method for producing a nerve cell sheet according to claim 3, wherein the concentration of at least one of the SDF1 and MCP-1 in the culture solution is 0.5 ng / mL to 30 ng / mL.   The method for producing a nerve cell sheet according to claim 3 or 4, wherein the sonic hedgehog inhibitor comprises at least one selected from the group consisting of cyclopamine, gerbin and tomatidine. The method for producing a nerve cell sheet according to any one of claims 3 to 5, wherein a concentration of the sonic hedgehog inhibitor in a culture solution is 2.0 × 10 ⁻⁷ μM to 3.0 μM.   The said differentiation-inducing agent includes at least one selected from the group consisting of retinoic acid, noggin, fibroblast growth factor (FGF) and nerve cell growth factor (NGF). The manufacturing method of the nerve cell sheet | seat of claim | item.   The progenitor cell is at least one selected from the group consisting of embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells, and neural progenitor cells. A method for producing a nerve cell sheet according to claim 1.   A sheet material for transplantation comprising the nerve cell sheet according to claim 1 or 2.