JP6399734B2 - Transdifferentiation control method and substrate - Google Patents

Description

  The present invention relates to a transdifferentiation control method and a substrate.

It is known to control cell differentiation, survival, proliferation, immune activity control, and the like with chemical factors in the liquid (see, for example, Reference 1).
Reference 1: Stella Pearson, Patrycja Sroczynska, Georges Lacaud and Valerie Kouskoff (2008)
"The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF, Development, 135, 1525-1535

  However, in the case where cell differentiation or the like is controlled using a chemical factor in the liquid, it is difficult to control because the chemical factor has diffusibility. In particular, it is difficult to control diffusive chemical factors in vivo. Furthermore, when cells to be controlled are transplanted into a living body, the remaining chemical factors may adversely affect the living body.

  In the first aspect of the present invention, there is provided a cell transdifferentiation control method, which is obtained by differentiating or transforming an original cell type having either one of differentiation ability and transformation ability. Preparing a substrate having a concavo-convex shape for inducing at least one of a stress fiber, a leaf-like temporary foot, and a thread-like temporary foot of an original cell based on a combination of a target cell type and a target cell; Placing the original cell on the cell and inducing at least one of the original cell stress fiber, leaf-like temporary foot, and filamentous temporary foot according to the concavo-convex shape to differentiate or transform into the target cell; Is provided.

  In the second aspect of the present invention, there is provided a substrate for controlling the shape of a cell, the substrate main body being provided on the substrate main body, and at least one of a cell stress fiber, a leaf-like temporary foot, and a thread-like temporary foot. And a concavo-convex shape for inducing.

  It should be noted that the above summary of the invention does not enumerate all the necessary features of the present invention. In addition, a sub-combination of these feature groups can also be an invention.

It is a schematic perspective view of the board | substrate 100 as an example of embodiment. It is the expansion perspective view which expanded a part of uneven | corrugated shape 104. FIG. It is a top view of the uneven | corrugated shape 104. FIG. It is a top view which shows the other uneven | corrugated shape 105. FIG. 2 is a perspective view of a petri dish 150. FIG. 4 is a perspective view of a well plate 160. FIG. FIG. 6 is a superimposed image when Example 3 is used. FIG. It is a superposition image at the time of using Example 4. The correlation between the uneven shape and the shape of the temporary foot is shown. (A) And (b) is a superposition image at the time of using Example 7. FIG. (C) and (d) are superimposed images when Example 8 is used. An example of a leaf-like temporary foot is shown conceptually. The correlation between the uneven shape and the shape of the temporary foot is shown. The correlation with uneven | corrugated shape and the position of a cell main body is shown. It is a superposition image at the time of using Example 11. FIG. It is a superposition image at the time of using Example 12. FIG.

  Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.

  FIG. 1 is a schematic perspective view of a substrate 100 as an example of an embodiment. The substrate 100 holds cells and promotes cell differentiation, transformation, culture, and the like. Further, the substrate 100 is used for observing the held cells with a microscope or the like.

  The substrate 100 includes a flat substrate body 102 having a length and width of several centimeters and a thickness of 0.17 mm, and a concavo-convex shape 104 disposed on one surface of the substrate body 102. In the example of FIG. 1, the concavo-convex shape 104 is provided on a part of one surface of the substrate body 102, but instead, it may be provided on the entire one surface.

  FIG. 2 is an enlarged perspective view in which a part of the concavo-convex shape 104 is enlarged, and FIG. 3 is a plan view thereof. The uneven shape 104 includes a plurality of columnar bodies 110 protruding from one surface of the substrate body 102. A gap 120 is formed between the plurality of columnar bodies 110.

  2 and 3, the columnar body 110 is a quadrangular column having a height h, and the upper surface 112 is a square having a width w. In addition, a plurality of columnar bodies 110 having the same shape are two-dimensionally arranged with gaps 120 having a certain size s. The height h, width w, and gap size s are all on the order of μm.

  The inventors have found that the width w and the gap size s control the shape of the cells. Based on this knowledge, a width w and a gap size s are set in the substrate 100 in order to control the cells to a desired shape.

  The concavo-convex shape 104 induces at least one of a stress fiber of cells, a leaf-like temporary foot, and a thread-like temporary foot. When the cell stress fiber is induced, the width w is preferably 3 μm to 10 μm. Further, when the cell temporary foot is induced, the gap size s is preferably 3 μm to 10 μm. In particular, when guiding a leaf-like temporary foot, the size s of the gap is preferably 5 μm to 10 μm. On the other hand, when the thread-like temporary leg is guided, the gap size s is preferably 3 μm to 5 μm.

  The substrate 100 is formed of glass, silicon, resin, or the like. In the case where the substrate 100 is formed of silicon, the uneven shape 104 is integrally provided on the substrate body 102 by using, for example, photolithography and etching. In the case where the substrate 100 is formed of a resin, a mold is formed using photolithography and etching, and the substrate body 102 and the concavo-convex shape 104 are integrally formed by injecting the resin into the mold and curing. Formed. Examples of the resin include polydimethylsiloxane (PDMS) and polylactic acid. Furthermore, the substrate 100 is preferably formed of a transparent material. Thereby, the cells of the substrate 100 can be easily observed with a microscope or the like.

  FIG. 4 is a plan view showing another uneven shape 105. 4, the same components as those in FIGS. 1 to 3 are denoted by the same reference numerals, and description thereof is omitted.

  In the concavo-convex shape 105 of FIG. 4, a plurality of columnar bodies 110 are arranged so that a main gap 122 and a sub gap 124 having a size different from the main gap 122 are formed. In the example of FIG. 4, the main gap size s1 is larger than the sub-gap size s2.

  The main gap 122 has a size s1 of 5 μm to 25 μm, and accommodates the cell body according to the size s1. On the other hand, the sub-gap 124 has a size s2 of 3 μm to 5 μm, and guides the filamentous temporary foot of the cell with the size s2. Alternatively, the sub-gap 124 may have a size s2 of 5 μm to 10 μm, and the cell-like temporary foot may be induced by the size s2. Further, the sub-gap 124 having a size s2 of 3 μm to 5 μm and the sub-gap 124 having a size s2 of 5 μm to 10 μm are mixed so as to induce the filamentous and lamellipodia to one cell, respectively. May be.

  Moreover, although the columnar body of FIGS. 1 to 4 is a quadrangular column, the shape of the columnar body is not limited to this. As other examples, a pentagonal prism or more polygonal column, a triangular column, a cylinder, a semi-cylinder, or the like may be used, and columnar bodies having different shapes may be mixed. Furthermore, columnar bodies having different heights may be mixed.

  FIG. 5 is a perspective view of the petri dish 150. The petri dish 150 has a circular bottom surface 154 and a cylindrical side wall 152 surrounding the outer periphery of the bottom surface 154. The concave / convex shape 104 shown in FIGS. 2 and 3 is formed on the bottom surface 154. The uneven shape 104 may be provided integrally with the bottom surface 154 and the substrate body 102 of the substrate 100 may be omitted, or the substrate 100 may be formed separately from the bottom surface 154 and attached to the bottom surface 154. Instead of the concavo-convex shape 104, the other concavo-convex shape 105 or the like may be provided.

  FIG. 6 is a perspective view of the well plate 160. The well plate 160 has a rectangular bottom surface 166 and a flat outer wall 162 surrounding the outer periphery of the bottom surface 166. The well plate 160 further has a plurality of cylindrical inner walls 164 surrounding the bottom surface 166 with a plurality of spaced regions. An uneven shape 104 is formed in a region surrounded by the inner wall 164 on the bottom surface 166. The uneven shape 104 may be provided integrally with the bottom surface 166, or may be formed separately from the bottom surface 166 and attached to the bottom surface 166. Instead of the concavo-convex shape 104, the other concavo-convex shape 105 or the like may be provided.

  In FIG. 6, six regions are provided, and the concavo-convex shape 104 is provided in all of the six regions. Instead of this, six or less regions or six or more regions may be provided, or the uneven shape 104 may not be provided in any of these regions. Moreover, uneven | corrugated shape 104,105 etc. of a different shape may be provided between those area | regions.

  The uneven shapes 104, 105 and the like may be further provided on the surface of the tissue regeneration scaffold. The uneven shapes 104, 105 and the like may be further provided on the surface of an implantable device, a cell function control chip for regenerative medicine, or the like.

(Example 1 to Example 4)
The substrate 100 of Examples 1 to 4 having the uneven shape 104 shown in FIGS. 1 to 3 and having the width w, the gap size s, and the height h shown in Table 1 was produced. The substrates 100 of Examples 1 to 4 were formed by forming a protective film pattern on a silicon wafer by photolithography and using deep reactive ion etching (DRIE). The surface of the concavo-convex shape 104 was coated with fibrectin (20 μg / ml, BD).

  Fibroblasts (Swiss / 3T3) were seeded on the upper surface 112 of the columnar body 110 of Examples 1 to 4, and the intracellular distribution of the actin cytoskeleton was observed. In addition to forming a stress fiber, the actin cytoskeleton is also a protein lining the cell surface layer immediately below the cell membrane, so that the cell shape can be identified by observing the intracellular distribution of the actin cytoskeleton.

  In this observation, in order to visualize the actin cytoskeleton, cells were fixed with 4% paraformaldehyde, and the actin cytoskeleton was fluorescently stained with Alexa Fluor 488 phalloidin (Invitrogen). Using a confocal laser microscope with a 60 × objective lens, a plurality of cross-sectional images of the fluorescently stained actin cytoskeleton were obtained while changing the focal position. A superimposed image was generated by superimposing the plurality of cross-sectional images.

  FIG. 7 is a superimposed image when Example 3 is used. FIG. 8 is a superimposed image when Example 4 is used.

  As can be seen from these drawings, the stress fiber tends to be strongly localized around the columnar body 110. Stress fibers were also localized in the columnar bodies 110 of Examples 1 and 2. It is known that a stress fiber is formed by activation of a Rho / ROCK pathway. Therefore, it is presumed that the columnar body 110 activates the Rho / ROCK pathway.

  From these figures, it can be seen that the temporary foot protrudes into the gap. When paying attention to the extension of the temporary foot in the direction along the surface, there is a tendency that the leaf-like temporary foot-like structure extends predominantly with respect to the wide gap as in Example 4 rather than the narrow gap as in Example 3. .

  FIG. 9 shows the correlation between the uneven shape and the shape of the temporary foot in Examples 1 to 4. Here, as the shape of the temporary foot, the width of the outer periphery of the temporary foot was measured as shown by the white arrow in FIG. As can be seen from FIG. 9, the fibroblasts formed a leaf-like temporary foot into the gap having a size s of 10 μm regardless of the width w of the columnar body 110. In the case where the width of the temporary foot was wide, a width exceeding 10 μm was observed. A leaf-like temporary foot was formed in a gap having a size s of 5 μm regardless of the width w of the columnar body 110. Most provisional foot widths were less than 5 μm.

  It is known that the lamellipodia are formed by activation of the Rac pathway. Thus, the gap is presumed to activate the Rac pathway.

(Example 5 to Example 10)
Substrates 100 of Examples 5 to 10 having the uneven shape 104 shown in FIGS. 1 to 3 and having the width w, the gap size s, and the height h shown in Table 2 were produced. The templates of the substrates 100 of Examples 5 to 10 were formed by photolithography using a resist SU-8 manufactured by Microchem. Further, PDMS was prepared by mixing PDMSSylgard184 of Dow-Corning and a curing agent at a volume ratio of 10: 1. The PDMS was spin coated on a mold and then cured at room temperature for 2 days. The substrate 100 was formed by peeling PDMS from the mold. The surface of the concavo-convex shape 104 of the substrate 100 was coated with fibronectin (20 μg / ml, BD).

  Fibroblasts (Swiss / 3T3) were seeded in the gaps 120 of the columnar bodies 110 of Examples 5 to 10, and the intracellular distribution of the actin cytoskeleton was observed. The observation method was the same as in Examples 1 to 4 above.

  FIGS. 10A and 10B are superimposed images when Example 7 is used. 10C and 10D are superimposed images when Example 8 is used. The white circles in the figure indicate the characteristic provisional foot shapes induced by the respective structures.

  As can be seen from FIGS. 10A and 10B, in Example 7 in which the size s of the gap is 3 μm, one thin temporary leg is formed in the gap. When this was confirmed with a 3D reconstructed image, it was a thread-like temporary foot with no spread in the height direction.

  On the other hand, as can be seen from FIGS. 10 (c) and 10 (d), in Example 8 in which the gap size s is 5 μm, two temporary feet are formed on both side surfaces of the gap. A temporary foot was formed to crosslink When this was confirmed with a 3D reconstructed image, it was a leaf-like temporary foot having a spread in the height direction.

  FIG. 11 conceptually shows an example of a leaf-like temporary foot. FIG. 12 shows the correlation between the uneven shape and the shape of the temporary foot in Examples 7 to 10.

  As shown in FIG. 11, among the temporary feet 130, the leaf-shaped temporary feet were bonded to the side surfaces of the columnar body 110 and the spread a in the height direction was 3 μm or more. In addition, a thread-like temporary foot that is bonded to the side surface of the columnar body 110 and has a height-direction spread a smaller than 3 μm and a thread-like temporary foot that is not bonded to the side surface of the columnar body 110 were evaluated.

  As a result, the ratio of temporary foot formation as shown in FIG. 12 was obtained. Here, the ratio of pseudopodia formation was an average value obtained by averaging the ratios of the pseudopodia formed in one cell to those of the lamellae and the filopodia. For Example 7, the average value of 4 cells was used, and for Example 8, the average value of 6 cells. For Example 9, the average value of 9 cells was used, and for Example 10, the average value of 8 cells.

  As is apparent from FIG. 12, in Example 7 in which the gap size s is 3 μm, the thread-like temporary foot is 95% or more, and in Example 9 in which the gap size s is 4 μm, the thread-like temporary foot is about 90%. It was a ratio. On the other hand, in Example 8 in which the gap size s was 5 μm and in Example 10 in which the gap size s was 7 μm, the number of leaf-like temporary feet was about half.

  As shown in Examples 1 to 10 above, in order to induce a leaf-like temporary foot, the gap size s is preferably 5 μm to 10 μm. Thereby, the pathway of Rac is more activated.

  Further, it is known that the filamentous temporary foot is formed by the activation of the cdc42 pathway. Therefore, it is estimated that a gap with a size s of 3 μm to 4 μm activates the cdc42 pathway.

  FIG. 13 shows the correlation between the uneven shape and the position of the cell body in Examples 5 to 8. Regarding the plurality of cells in Examples 5 to 8, the respective cell bodies are “all present in the gap 120”, “some are present in the gap 120 and some are present on the upper surface 112 of the columnar body 110”. The numbers were counted by distinguishing them from “all exist on the upper surface 112”.

  As a result, as shown in FIG. 13, when the width w of the columnar body 110 is 7 μm and the gap size s is 3 μm or more, and when the width W is 10 μm and the gap size s is 5 μm or more, For more than% cells, all or part of the cell body was in the gap 120. In addition, in all cases where the width w was 7 μm and 10 μm, all of the cell bodies were present in the gap 120 for 60% or more of the cells when the gap size s was 3 μm or more.

(Example 11 and Example 12)
Substrates 100 of Examples 11 and 12 having the uneven shape 105 shown in FIG. 4 and having the width w, the main gap size s1, the subgap size s2 and the height h shown in Table 2 were produced. The preparation method was in accordance with Examples 1 to 4.

  Fibroblasts (Swiss / 3T3) were seeded in the main gap 122 of the columnar body 110 of Examples 11 and 12, and the intracellular distribution of the actin cytoskeleton was observed. The observation method was the same as in Examples 1 to 4 above.

  FIG. 14 is a superimposed image when Example 11 is used. As shown by the arrowhead and the arc portion in FIG. 14, the leaf-shaped temporary foot was induced predominantly. The arrowhead indicates a temporary foot having a spread in the height direction. The circular arc indicates a temporary foot having a spread in a direction along the surface.

  FIG. 15 is a superimposed image when Example 12 is used. As shown by the arrowheads in FIG. 15, a small number of thread-like temporary feet were formed, but no leaf-like temporary feet were formed.

  Therefore, in order to accommodate the entire cell body, suppress the formation of stress fibers, and effectively perform the temporary foot shape operation, the size s1 of the gap 120 and the size s1 of the main gap 122 are both 5 μm or more. It is preferable that Furthermore, it is more preferable to apply poly-lysine-g-poly (ethylene glycol) or the like that inhibits cell adhesion to the upper surface 112 of the columnar body 110. In particular, the size s1 of the main gap 122 is more preferably about 25 μm or less. Furthermore, the size s2 of the sub-gap 124 is preferably 5 μm or more for growing the leaf-like temporary foot, and 5 μm or less is preferred for growing the thread-like temporary foot.

  As described above, according to the present embodiment, the shape of the cell can be controlled by the micro-order uneven shape 104 or the like. As a result, the cells can be stably maintained or grown for a long period of about several weeks. In addition, since the physical interaction of the concavo-convex shape 104 is utilized, an exogenous chemical factor does not remain in the cell tissue and can be made biochemically non-invasive.

Table 4 shows an example of combinations of original cells and target cells according to other embodiments. In this embodiment, the substrate 100 is used to control the differentiation or transformation from the original cell to the target cell.

  First, a combination of an original cell type having either one of differentiation ability and transformation ability and a target cell type obtained by differentiating or transforming the original cell is specified. As shown in Table 4, an example of the original cell type having differentiation potential is a mesenchymal stem cell. Examples of cell types of interest in that case are smooth muscle cells, chondrocytes, adipocytes and osteoblasts. An example of the type of the original cell having transformation ability is fibroblast. Examples of cell types of interest in that case are smooth muscle cells, chondrocytes, adipocytes and osteoblasts.

  Based on the above combination, a substrate 100 having a concavo-convex shape 104 or the like for guiding at least one of the stress fiber of the original cell, the leaf-like temporary foot and the thread-like temporary foot is prepared. Here, by the above combination, it is specified which one of the stress fiber, the leaf-like temporary foot, and the thread-like temporary foot should be preferentially induced from the original cells, and the substrate 100 on which they are preferentially induced is prepared.

  The original cells are placed on the substrate 100, and the at least one of the stress fibers, the leaf-like limbs and the thread-like limbs of the original cells is induced by the concavo-convex shape 104 to differentiate into the target cells. Or transform.

  As shown in Examples 1 to 10 above, by selecting the uneven shape 104 of the substrate 100 for the fibroblast, each of the stress fiber, the leaf-like temporary foot and the thread-like temporary foot is selectively induced. Furthermore, fibroblasts are known to be transformed into smooth muscle cells, chondrocytes, adipocytes and osteoblasts. In contrast, mesenchymal stem cells are differentiated into smooth muscle cells when induced predominantly with lamellipodia (eg, reported by Gao et al. 2010 Stem Cells), and differentiated into osteoblasts when stress fiber is induced predominantly. (Eg, reported by McBeath et al. 2004 Dev. Cell).

  According to the above, the concavo-convex shape 104 corresponding to the shape to be induced is determined based on the differentiation of the mesenchymal stem cell into the smooth cell, the chondrocyte, the fat cell and the osteoblast from the original cell. By specifying the substrate 100 to be included and controlling the shape of the cell using the substrate 100, it is considered that the target cell can be differentiated. Similarly, it has a concavo-convex shape 104 or the like corresponding to the shape to be induced, based on whether fibroblasts are transformed into smooth muscle cells, chondrocytes, adipocytes, or osteoblasts. It is considered that a target cell can be transformed by specifying the substrate 100 and controlling the shape of the cell using the substrate 100. In addition to using the substrate 100 having the concavo-convex shape 104 or the like corresponding to the shape to be guided, it is preferable to perform the additional processing shown in Table 4 above.

  Other examples of cells having differentiation ability include embryonic stem cells, induced pluripotent stem cells, endoderm stem cells, mesoderm stem cells, ectoderm stem cells, hematopoietic stem cells, vascular endothelial progenitor cells and neural stem cells. Using these as original cells, the substrate 100 having the concavo-convex shape 104 corresponding to the shape to be induced can be identified according to the target cell to be differentiated, and can be differentiated into the target cell using the substrate 100. it is conceivable that.

  As described above, according to the present embodiment, differentiation and transformation can be controlled using the substrate 100 having the micro-order uneven shape 104 or the like. Since the physical interaction of the concavo-convex shape 104 is used, foreign chemical factors do not remain in the cell tissue and can be made biochemically non-invasive.

  As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

  The order of execution of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior to”. It should be noted that the output can be realized in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for convenience, it means that it is essential to carry out in this order. It is not a thing.

  100 substrate, 102 substrate main body, 104 uneven shape, 105 uneven shape, 110 columnar body, 112 upper surface, 120 gap, 122 main gap, 124 sub-gap, 130 temporary leg, 150 petri dish, 152 sidewall, 154 bottom surface, 160 well plate, 162 outer side wall, 164 inner side wall, 166 bottom surface

Claims (7)

A method for predominantly inducing mesenchymal stem cells or fibropodia lamellipodia ,
In the method, the uneven shape of the columnar body is formed on a substrate arranged in a rectangular lattice pattern by providing a main gap and a sub-gap having a width different from the main gap in a uniaxial direction and an axial direction different from the uniaxial direction. A method of placing mesenchymal stem cells or fibroblasts ,
The width of the main gap is 5 μm to 10 μm,
The width of the sub gap is smaller than the width of the main gap within a range of 5 μm to 10 μm ,
The side of the columnar body is coated with a cell adhesive material, the top surface that is coated with a cell non-adhesive substance, method. A method for preferentially inducing filamentous pseudopodia of mesenchymal stem cells or fibroblasts ,
In the method, the uneven shape of the columnar body is formed on a substrate arranged in a rectangular lattice pattern by providing a main gap and a sub-gap having a width different from the main gap in a uniaxial direction and an axial direction different from the uniaxial direction. A method of placing mesenchymal stem cells or fibroblasts ,
The width of the main gap is 5 μm to 10 μm,
The width of the sub-gap Ri 5μm below der from 3 [mu] m,
The side of the columnar body is coated with a cell adhesive material, the top surface that is coated with a cell non-adhesive substance, method. A substrate for predominantly inducing mesenchymal stem cells or fibroblasts of foliate pseudopods ,
On the substrate, the concavo-convex shape of the columnar body is arranged in a rectangular lattice pattern with a main gap and a sub gap having a width different from the main gap in a uniaxial direction and an axial direction different from the uniaxial direction, respectively.
The width of the main gap is 5 μm to 10 μm,
The width of the sub gap is smaller than the width of the main gap within a range of 5 μm to 10 μm ,
The side of the columnar body is coated with a cell adhesive material, the top surface that is coated with a cell non-adhesive material, the substrate. A substrate for preferentially inducing filamentous pseudopods of mesenchymal stem cells or fibroblasts ,
On the substrate, the concavo-convex shape of the columnar body is arranged in a rectangular lattice pattern with a main gap and a sub gap having a width different from the main gap in a uniaxial direction and an axial direction different from the uniaxial direction, respectively.
The width of the main gap is 5 μm to 10 μm,
The width of the sub-gap Ri 5μm below der from 3 [mu] m,
The side of the columnar body is coated with a cell adhesive material, the top surface that is coated with a cell non-adhesive material, the substrate. The substrate according to claim 3 or 4, which is integrally formed of glass, silicon, or resin. A petri dish in which the substrate according to claim 3 is disposed on a bottom surface. A well plate in which the substrate according to claim 3 is disposed on a bottom surface.