JP2015228797A - Method for producing stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing stem cells from stromal cells.SOLUTION: The method for producing stem cells, the method comprising a step of culturing stromal cells (mesenchymal stromal cells), rotating and/or shaking a culture vessel, is a method for producing differentiated cells, the method comprising a step of culturing the stem cells, which are obtained after the stromal cell culturing step, in a culture medium for inducing differentiation. In the method, the differentiated cells are selected from a group consisting of hepatocyte, adipocyte, osteocyte and neurocyte.

Description

  The present invention relates to a method for producing stem cells from stromal cells, for example.

  In recent years, research on stem cells such as iPS cells has been promoted in the field of regenerative medicine. In this research, a method for producing stem cells by applying stress to cells such as adipose tissue-derived cells is known.

  For example, Patent Document 1 uses a stress caused by low oxygen culture to separate adipose tissue-derived cells attached to a solid phase into cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase. Disclosed is a method for producing adipose tissue-derived somatic stem cells, which comprises a step of low-oxygen culturing of detached cells. Patent Document 1 discloses that the obtained adipose tissue-derived somatic stem cells have the ability to differentiate into adipose tissue, bone tissue or nerve cells.

  Patent Document 2 discloses a method for primary culture of mesenchymal stem cells, comprising culturing mesenchymal stem cells derived from human adipose tissue in a medium not containing serum using stress due to culture using a serum-free medium. Is disclosed.

  However, a method for producing stem cells simply and in large quantities from cells such as adipose tissue-derived cells due to physical stress has not been known.

JP 2014-30423 JP 2012-157263 A

  In view of the above circumstances, an object of the present invention is to provide a method for easily producing a large amount of stem cells from cells such as adipose tissue-derived cells by physical stress.

  As a result of diligent research to solve the above problems, stromal cells such as adipose tissue-derived stromal cells that originally adhere and proliferate are contained in a culture vessel (medium) containing the stromal cells so that they cannot adhere. The inventors have found that stem cells can be obtained by culturing under the stress of rotating (rotating), and have completed the present invention. That is, the present invention includes the following.

(1) A method for producing stem cells, comprising a step of culturing stromal cells in the incubator while rotating and / or vibrating the incubator.
(2) The method according to (1), wherein the stromal cells are mesenchymal stromal cells.
(3) The method according to (2), wherein the mesenchymal stromal cells are adipose tissue-derived stromal cells.
(4) A stem cell produced by the method according to any one of (1) to (3).
(5) A method for producing differentiated cells, comprising a step of culturing the obtained stem cells in a differentiation-inducing medium after the stromal cell culturing step in the method according to any one of (1) to (3).
(6) The method according to (5), wherein the differentiated cells are selected from the group consisting of hepatocytes, adipocytes, bone cells and nerve cells.

  According to the method for producing stem cells according to the present invention, stem cells can be produced easily and in large quantities by physical stress.

It is a graph which shows the colony size and dispersion degree of rota-ADSC which checked and evaluated with the microscope. When the colonies were placed on the plate, the colony size could be confirmed more reliably with imageJ. It is a photograph showing rota-ADSC (P9 → P10) 48 hours after rotation suspension (before adhesion, 2nd day). It is a photograph which shows rota-ADSC (P10) 24 hours (3rd day) after adhesion | attachment to a culture dish. It is a figure which shows the determination method of colony size distribution of rota-ADSC. ADSC (P10) was used at a concentration of 175000 cells / mL and a final volume of 17 mL. After 48 hours, rota-ADSC images were taken, and hemocytometer images with the same size and focus were taken and overlapped. The obtained image was used for setting the scale (0.365 pixel / μm) of ImageJ. FIG. 5 is a diagram in which ImageJ (http://imagej.nih.gov/ij/) is used for the processing and analysis of the image shown in FIG. 4 to determine the number and surface area of each colony. The number assigned to each colony is the number in the raw data. It is a graph which shows colony size distribution and cumulative capacity of rota-ADSC. Graph 1 shows the size distribution of rota-ADSC colonies. Each column shows colony counts (left y-axis) over a range of diameters. The right y-axis (Graph 1) shows the total volume obtained by summing all colony volumes in a specific diameter range (x-axis). The diameter value used for the total volume is not the raw value obtained by imageJ, but the value obtained by “bin center” (previously obtained from the graph plot) multiplied by the count. The x-axis in graph 1 is common to both y-axes. After induction of rota-ADSC, the majority of cells were present in colonies with diameters ranging from 50-200 μm. The number of cells in each colony is not certain and the density of the colonies is not known and depends on the colony size. By estimating that the volume occupied by a single cell was constant (1 cell / 524 μm ³ ), the number of cells obtained per colony could be predicted (Graph 2). It is a time-lapse image of GFP-rota-ADSC (P9) after plating on a culture dish. It is a graph which shows the qRT-PCR result regarding the expression of each gene in rota-ADSC and ADSC. Positive expression of Nanog, Sox2, Gata6, Klf4, Eras, Esg1 and Rex1 was confirmed by qRT-PCR in both rota-ADSC colonies (P9 and P16). In naive ADSC (P8), only KLF4, Par4 and c-Myc were expressed. In the rota-ADSC population, capacity was indeed increased (up-regulation of Eras, Esg1 and Rex1), and self-replicating ability was maintained. The data potentially indicate that there is no difference in induction to rota-ADSC colonies in new (P7-10) or old (P15-17) passage ADSCs. Both old and young ADSCs are likely to show similar patterns. It is a graph which shows the cytoskeletal component in rota-ADSC and naive ADSC. Reduced vinculin expression emphasized decreased cell binding and altered cell migration (Critchley, DR, Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion. Biochemical Society Transactions, 2004. 32: p. 831-836) . On the other hand, a moderate increase in profilin 1 promotes the disappearance of cytoplasmic actin filaments, and possibly the resulting accumulation of these filaments is located at the outer cell edge and can form a thick cell cortex margin (Roy , P. and K. Jacobson, Overexpression of profilin reduces the migration of invasive breast cancer cells. Cell Motility and the Cytoskeleton, 2004. 57 (2): p. 84-95). CDH1 was down-regulated and a slight increase in overall β-catenin expression and fibronectin expression was recognized. It is a graph which shows the expression of the surface marker in rota-ADSC and naive ADSC. The percentage of positive cells was analyzed after elimination of debris and dead cells (using 7-AAD). In order to minimize non-specific staining, FcR blocking reagent was used prior to antibody staining. A graph showing all the major genes involved in 25 biological processes. The x-axis shows the number of occurrences in a particular process with a single gene. It is a figure which shows the differentiation (test 1) from the ADSC cluster to the cell which expresses albumin positively. It is a figure which shows the differentiation (test 2) to the hepatocyte of rota-ADSC. The differentiation method shown in Example 2 shows the results shown in the graphs and photographs. Albumin expression was present on day 15 and Hnf1b expression was increased 10-fold in differentiated hepatocytes after rota-ADSC cluster formation. . It is a graph which shows the differentiation test (control) from naive ADSC to a hepatocyte-like cell. Differentiation from naive ADSCs into hepatocyte-like cells did not occur. Neither albumin nor afp was expressed. It is a figure which shows the adipocyte differentiation from rota-ADSC. In differentiated cells, Pparg and Tfap2a were expressed 20-30 times higher than the starting rota-ADSC cells or naive ADSCs. This result confirmed the differentiation form into fat cells seen in the image. FIG. 16 is a photograph showing the differentiation of rota-ADSC into adipocytes, supplementing FIG. 15. It is a figure which shows the bone cell differentiation from rota-ADSC and naive ADSC. With respect to bone cell differentiation, there was no difference in the differentiation potential of the two populations investigated. All checked genes were up-regulated. It is a figure which shows the nerve differentiation from rota-ADSC. Similar to motor neuron precursors and motor neurons, neurogenin-2 was expressed after two-step differentiation. Gfap expression indicates the formation of glial fibrillary acidic protein normally found in astrocytes.

  The method for producing a stem cell according to the present invention is such that the stromal cells that are originally attached and proliferate cannot be adhered under non-adhesive culture stress, that is, while rotating and / or vibrating the incubator (medium). This is a method for producing stem cells by culturing stromal cells in an incubator. According to the method for producing stem cells according to the present invention, cells having anoikis resistance are selected from stromal cells under physical stress of rotating and / or vibrating the incubator, and via cell-cell interaction. By inducing cell clustering, stromal cell-derived stem cells can be obtained.

  Here, examples of stromal cells include adipose tissue-derived stromal cells, bone marrow, umbilical cord stromal cells, and mesenchymal stromal cells present in all tissues. In addition, the stromal cells may be derived from mammals such as humans, monkeys, mice, rats, rabbits, cows, and pigs.

In the method for producing stem cells according to the present invention, stromal cells are cultured in the medium in the incubator while rotating and / or vibrating the incubator. As the incubator, for example, a container such as a falcon tube can be used. The medium may be usually used for stromal cell culture. In the case of adipose tissue-derived stromal cells, for example, 100 units / ml penicillin, 100 microgram / ml streptomycin, 250 ng / Medium (eg, DMEM / F12 medium) containing ml of amphotericin B, 2 mmol / L L-Alanyl-L-Glutamine and 2-20% (preferably 2-10%) serum (eg, FBS) Is mentioned. The concentration of stromal cells in the culture is, for example, 1 × 10 ⁴ to 1 × 10 ⁶ cells / ml, preferably 1.5 × 10 ^{5 to} 2.0 × 10 ⁵ cells / ml.

  In the rotation treatment, for example, a rotator such as Miltenyi Biotec MX001 Macsmix Tube Rotator is used to rotate the incubator at a rotation speed of 4 to 12 rpm, preferably 8 to 12 rpm so that stromal cells do not adhere to the incubator. The rotation treatment (cultivation) time may be a time during which colonies do not aggregate into a large structure, and is, for example, 12 to 72 hours, preferably 36 to 60 hours.

  On the other hand, the vibration treatment is performed, for example, in a shaking incubator at a setting of 50 to 220 rpm for the same time as the above rotation treatment.

  After the rotation / vibration treatment (culture), the cells are isolated by centrifugation or the like, seeded again in the medium, and adhered cells (or cell colonies) can be obtained as stem cells.

  Evaluation of whether or not the obtained stem cells have self-renewal ability and differentiation ability can be performed using, for example, the expression of undifferentiated cell markers (for example, Nanog, Sox2, Gata6, Klf4, Eras, Esg1, Rex1, etc.) as an index. it can.

  The stem cells thus obtained can be differentiated into cells of three germ layers (endoderm, mesoderm, and ectoderm) by culturing in a differentiation-inducing medium. Examples of differentiated cells include hepatocytes, adipocytes, bone cells, nerve cells and the like.

  Examples of medium for inducing differentiation from stem cells to hepatocytes include 2-6% FBS, 2 mM glutamine, 100 units / ml penicillin, 100 microgram / ml streptomycin, 250 ng / ml amphotericin B, 250 nM dexamethasone. (Dex), CC-4182 HCM SingleQuots Kit (Lonza) (this kit contains ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor (rhEGF) and GA-1000; And a medium (for example, DMEM medium) containing 100 to 300 ng / ml (preferably 200 ng / ml) of hepatocyte growth factor (HGF). Moreover, it is preferable to culture | cultivate with a laminin coating plate. Examples of the culture time include 13 to 18 days.

  Differentiation from stem cells to hepatocytes can be evaluated using the expression of hepatocyte markers (eg, albumin) as an index.

  As a differentiation induction medium from stem cells to adipocytes, for example, 10% FBS, 100 units / ml penicillin, 100 microgram / ml streptomycin sulfate, 2 mM glutamine, 100x adipogenic supplement (hydrocortisone, isobutylmethylxanthine, A medium (for example, α-MEM medium) containing indomethacin). Examples of the culture time include 6 to 9 days.

  Differentiation from stem cells to adipocytes can be evaluated using, for example, the expression of adipocyte markers (eg, Pparg, Tfap2a) as an index.

  As a differentiation induction medium from stem cells to bone cells, for example, 10% FBS, 100 units / ml penicillin, 100 microgram / ml streptomycin sulfate, 2 mM glutamine, and 20x mOsteogenic supplement (ascorbate-phosphate, β- a medium containing glycerolphosphate and recombinant BMP-2 (for example, αMEM medium). Moreover, it is preferable to culture | cultivate with a laminin coating plate. Examples of the culture time include 17 to 23 days.

  Differentiation from stem cells into bone cells can be evaluated using, for example, the expression of bone cell markers (eg, Alpl, Runx2, Bglap, Col1a1) as an index.

  Differentiation from stem cells to neurons can be performed in two stages of culture steps. As the differentiation induction medium in the first culture step, for example, 50x B-27 supplement (cat # 17504044, Life Technologies), 100 microgram / ml kanamycin, 2 mM glutamine (Gln), 30 ng / ml basic Examples include a medium (eg, Neurobasal medium) containing fibroblast growth factor (bFGF) and 30 ng / ml EGF. Examples of the culture time for the first culture step include 7 to 9 days.

  Next, after the first culture step, the medium is changed and the second culture step is performed. Examples of the differentiation induction medium in the second culture step include a medium (for example, α-MEM medium) containing 2% FBS, 25 ng / ml bFGF, and 25 ng / ml brain-derived neurotrophic factor (BDNF). . Further, the culture in the second culture step is preferably performed on a laminin-coated plate. Examples of the culture time for the second culture step include 9 to 12 days.

  Differentiation from stem cells to nerve cells can be evaluated using, for example, the expression of a nerve cell marker (for example, Neurog2, Gfap) as an index.

  As described above, according to the method for producing a stem cell according to the present invention, a stem cell useful in the field of regenerative medicine and the like can be produced.

  For example, liver diseases such as hepatitis (alcoholic / nonalcoholic), fatty liver, cirrhosis, and liver cancer are rapidly increasing in Japan due to changes in lifestyle habits. In liver disease malignancy, preservation measures are ineffective and liver transplantation must be performed. However, in transplantation, the number of donors is small and rejection is a problem.

  Stem cells produced by the method for producing stem cells according to the present invention can be differentiated into hepatocytes. Therefore, according to the method for producing stem cells according to the present invention, stem cells are produced from patient-derived stromal cells, and further hepatocytes are differentiated from the obtained stem cells, whereby liver transplantation required as described above Hepatocytes for use in can be provided.

  Further, according to the method for producing stem cells according to the present invention, stem cells are produced from bovine stromal cells, and hepatocytes are differentiated from the obtained stem cells to provide an edible liver (lever). it can.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

  In the following examples and drawings to be referred to, “naive ADSC” is an adipose-derived stromal cell before induction into stem cells, and “rota-ADSC” is induction into stem cells. It is a later adipose tissue-derived stromal cell (stem cell).

  Penicillin and streptomycin (sulfate) may be collectively referred to as “P / S”, and penicillin, streptomycin (sulfate), and amphotericin B may be collectively referred to as “A / A (antibiotic-antimycotic)”. .

[Example 1] Induction of rota-ADSC cluster from naive ADSC and its characterization
1. Materials and methods
1-1. Extraction of mouse adipose tissue-derived stromal cells (ADSC) Mouse ADSCs were collected from the inguinal fat of sacrificed mice (C57BL / 6JCrj or BL / 6-GFP male mice).

  Lymph nodes were removed and the tissue was placed in PBS (1 ml in a 3 ml vial) and cut evenly with scissors. The fat in PBS was placed in a 50 mL falcon tube, 5 mL PBS was added and subjected to centrifugation at 1600 rpm for 5 minutes. This washing step was repeated one more time.

After centrifugation, the intermediate wash PBS was removed while avoiding aspiration of the supernatant and pellet. Then 5 ml of 0.15% w / v collagenase I solution was added and the suspension was placed in Miltenyi Biotec MX001 Macsmix Tube Rotator. The rotation (rotation) was carried out at a maximum speed of 12 rpm at 37 ° C. and 5% CO ₂ for 20 minutes. During rotation, the falcon cap was sealed with paraffin film.

  After rotation, 10 mL of medium (DMEM-F12) was added to the suspension to stop the collagenase treatment and suppress the enzyme reaction. The suspension was subjected to centrifugation at 1800 rpm for 15 minutes.

  After centrifugation, the supernatant and the suspended layer of lipid droplets were removed and the pellet was resuspended in 10 mL of medium. The suspension was filtered with a 100 μm cell strainer and centrifuged at 1800 rpm for 6 minutes. After the centrifugation, the supernatant was removed, the pellet was suspended in 4.5 mL of fresh medium, and the resulting cells were seeded in one 6 cm culture dish.

  Cells were detached by trypsinization and passaged several times. In this way, ADSC used for induction into stem cells (rota-ADSC) was passaged 7 to 10 times. The medium used was DMEM / F12 (500 mL) containing FBS (20%), A / A (5 mL) and glutamine (5 mL).

1-2. Rota-ADSC induction protocol Mouse rota-ADSC cells were produced by treating the naive ADSC obtained in Section 1-1. rota-ADSC colonies were obtained consistently regardless of the number of passages of ADSC (usually using cells passaged 7-9 times). Specifically, guidance from naive ADSC to rota-ADSC was performed according to the following protocol.

(1) ADSC was peeled off with trypsin / EDTA (0.05% w / v), and trypsin was neutralized by adding a medium. The total number of cells was then calculated.
(2) The mixture was centrifuged at 1600 rpm for 5 minutes, and the supernatant was aspirated.
(3) Resuspend the pellet in DMEM / F12 / penicillin (100 units / ml) / streptomycin (100 microgram / ml) / amphotericin B (250 ng / ml) / glutamine (Gln) (2 mM) / FBS 20% The cell concentration was 1 × 10 ^{5 to} 3 × 10 ⁵ cells / ml. The final volume ranged from 3-25 ml. When using a final volume of less than 6 ml, there was a high risk of forming a very large cell mass during the rotary suspension (FIG. 1). In addition, the size of colonies and the degree of size dispersion could be adjusted by modifying these three constraints.
(4) The Falcon tube containing the cells was sealed with 3 layers of parafilm and finally sealed with a layer of strong yellow tape. Also, ethanol was used to sterilize the falcon tube and parafilm at each stage. Thus, the top of the falcon tube was sterilized to avoid leakage and contamination during rotation.
(5) The Falcon tube was placed in a Miltenyi Biotec MX001 Macsmix Tube Rotator and rotated at a maximum speed of about 12 rpm for 36-66 hours (FIG. 1).
(6) By setting the minimum time of treatment to 36 hours, apoptosis was induced for all single cells that did not rearrange to colony formation. The maximum time is 66 hours, beyond which colonies tend to become larger structures and aggregate, and cells in the colony's inner core (especially those in larger colonies) are subject to low oxygen and nutrient intake. Due to it began to die.
(7) The Falcon tube was centrifuged at 1600 rpm for 5 minutes, the upper parafilm / yellow tape was removed, and the supernatant was aspirated.
(8) Resuspend the pellet in DMEM / F12 / A / A / Gln / FBS 20% and place the cells / colony on a plate with a surface area equivalent to 1/6 of the surface area occupied by the naive ADSC before spinning. Sowing.
(9) Viable cells / colony adhered to the plate within 8 hours. The remaining cells in suspension were discarded when the medium was changed.
(10) Information on characteristic evaluation of the obtained rota-ADSC is shown in FIGS.

2. Results and Discussion A spin / suspension method was found to stress mouse ADSCs, select cells with anoikis resistance, and induce cell clustering via cell-cell interactions. rota-ADSC colonies were obtained using a suspension / vibration system. The diameter of the obtained colonies could be adjusted and ranged from 45 μm to 180 μm. After 48 hours of suspension / vibration, many of the ADSCs that were not reorganized into colonies faced apoptosis and cells with an initial cell number of approximately 50-80% died during the procedure.

  Positive expression of Nanog, Sox2, Gata6, Klf4, Eras, Esg1 and Rex1, both in young passage rota-ADSC colonies and in many passages rota-ADSC colonies (passage 9 (P9) and P16, respectively) In qRT-PCR. In naive ADSC (P8), only KLF4, Par4 and c-Myc were expressed. In the rota-ADSC population, capacity was indeed increased (up-regulation of Eras, Esg1 and Rex1), and self-replicating ability was maintained. The data shown in FIG. 8 shows potentially no difference in induction to rota-ADSC colonies in the new (P7-10) or old (P15-17) passage ADSCs. Both old and young ADSCs appear to show similar patterns (Figure 8).

  In the suspension / vibration system, cells that were considered to have high ability and enhanced aggregation ability in the ADSC population were selected.

When the surface markers of rota-ADSC were checked and compared with those of naive ADSC, the most significant differences were seen in the following points (Figure 10):
(1) ↑ CD44-cell surface glycoproteins involved in cell-cell interactions, cell adhesion and migration; due to the production of collagen, fibronectin and laminin in colonies, an increase in CD44 is reasonable. CD44 gene transcription is activated in part by β-catenin and Wnt signaling.
(2) ↓ CD49b-checked to monitor adhesion behavior to extracellular matrix.
(3) ↓ CD73-decreased cell replication.
(4) ↓ CD34—proliferation down-regulation and undifferentiated state failure.
(5) ↓ Notch-1-apoptosis may induce Notch-1 down-regulation (which may stop differentiation).
(6) ↓ A decrease that may be due to the death of cells expressing CD95-apoptosis-related receptors.

  During the period of rotation, growth stopped. Down regulation of some surface markers was attributed to some ADSCs that faced apoptosis.

<Up-regulated path in rota-ADSC vs. naive ADSC>
We examined microarray gene expression of up-regulated genes and highlighted differences between naive ADSC and rota-ADSC.

  A total of 1513 genes were screened by microarray and systematically analyzed to extract biological significance.

  In rota-ADSC, MAP, JNK and p38 MAP kinase pathways were upregulated. The JNK and p38 MAP kinase pathways are induced by serum or reactive oxygen species. ERK5, PI3K-Akt signaling and TGF-β signaling pathways were also activated. This meant an increase in cell proliferation, inflammation and apoptosis / anti-apoptosis regulatory alerts and was also confirmed by activation of the Jak-STAT signaling pathway.

  Lysosomal glycosidase was overproduced in rota-ADSC. Regulation of the actin cytoskeleton facilitates pathways comparable to membrane remodeling and transport, pseudopod expansion, particle internalization, phagosome occlusion, and eventual phagosome-lysosome fusion, FcyR-mediated phagocytosis. Intracellular degradation of proteins is thought to switch from the ubiquitin-dependent process to the autophagy-lysosome pathway in rota-ADSC.

  Cytokine-cytokine receptor interactions have been upregulated, particularly with respect to the hematopoietin family. Furthermore, inflammatory cytokines were produced via toll-like receptor signaling pathways (TLR2, MD-2 and IFNAR2 receptors) and possibly induced by peptidoglycan, lipoprotein or lipopolysaccharide.

  In rota-ADSC, local adhesion was affected by cell interaction with extracellular matrix (ECM). This ECM was formed by collagen, laminin, vitronectin, secreted phosphoprotein-1 produced by the clustered cells themselves, and integrin was also overproduced. Motility was hampered by loss of substrate, and degradation of the actin cytoskeleton was a direct result, whereas the cytoskeleton was maintained in naive ADSCs. Cytokine-cytokine receptor interactions along with ECM-receptor interactions promoted the Wnt signaling pathway and activated JNK signaling. The result was significant cell survival and proliferation. In addition, phosphatidylinositol signaling partially prevented actin polymerization.

  The cytoskeleton changed during stress manipulation. In naive ADSCs, there are adhesive junctions, desmosomes, nactin and stress fibers, whereas genes expressed in rota-ADSC are responsible for the formation of filopodia and membranous pseudopods and all other types of binding. Emphasized regulation.

  In rota-ADSC, gonadotropin gene expression and secretion was active, and there was increased glucose cell uptake and upregulation of sphingolipid metabolism. The macromolecules produced were phosphoethanolamine and ceramide.

  Reduced vinculin expression emphasized decreased cell binding and altered cell migration (Critchley, DR, Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion. Biochemical Society Transactions, 2004. 32: p. 831-836) . On the other hand, a moderate increase in profilin 1 promotes the disappearance of cytoplasmic actin filaments, possibly resulting in the accumulation of these filaments in the extracellular margin at the thick cortical margin (Roy, P. and K. Jacobson, Overexpression of profilin reduces the migration of invasive breast cancer cells. Cell Motility and the Cytoskeleton, 2004. 57 (2): p. 84-95). CDH1 was down-regulated and a slight increase in overall β-catenin expression and fibronectin expression was recognized (FIG. 9).

List of up-regulated laminin and collagen related genes:
(1) Laminin α-1: the functional role of laminin α1 chain during cerebellar development and epidermal endoderm development (Kaestner, K., Development, Differentiation, and Disease of the Luminal Gastrointestinal Tract. 2010);
(2) Laminin, α4: LAMA4 regulates MMP3 expression (highly upregulated in rota-ADSC cluster), possibly via syndecan-4 (Fuerst, FC, et al., Matrix metalloproteinase 3 is regulated by the laminin LAMA4 in human osteoarthritis. 2011. p. A14);
(3) laminin γ1;
(4) Col2a1: involved in bone and cartilage differentiation;
(5) Col3a1: Vascular structure and intestinal development;
(6) Col5a3;
(7) Col15a1;
(8) Col22a1: An extracellular matrix protein that exists only in tissue binding (Koch, M., et al., A Novel Marker of Tissue Junctions, Collagen XXII. 2004. p. 22514-22521).

  Table 1 below shows 25 biological processes highlighting the analysis of rota-ADSC compared to naive ADSC. The program used was Analysis Wizard of DAVID Bioinformatics Resources 6.7 (Huang, DW, BT Sherman, and RA Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols, 2008. 4 (1) 44-57; Huang, DW, BT Sherman, and RA Lempicki, Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. 2009. p. 1-13).

  FIG. 11 also shows all the major genes involved in the 25 biological processes. The x-axis shows the number of occurrences in a particular process with a single gene.

[Example 2] Verification of pluripotency of rota-ADSC cells induced by direct differentiation into three germ layers
1-1. Rota-ADSC toward endoderm lineage
<Procedure for hepatocyte differentiation>
(1) According to the method described in Example 1, rota-ADSC colonies (P10) obtained by the suspension / vibration system of ADSC (P9) were seeded in 6 wells of a 24 laminin-coated well plate. Approximately 70000 cells per well were seeded, and 400 μl of the following predifferentiation medium was added per well and cultured for 15 days: 4% FBS, 2 mM glutamine (Gln), 100 units / ml penicillin, 100 microgram / ml streptomycin, 250 ng / ml amphotericin B, 250 nM dexamethasone (Dex), CC-4182 HCM SingleQuots Kit (Lonza) DMEM medium containing recombinant human epidermal growth factor (rhEGF) and GA-1000; use dose according to manufacturer's instructions) and 150 ng / ml recombinant mouse hepatocyte growth factor (rmHGF) .
(2) Medium exchange was performed on days 0, 1, 4, and 11.
(3) RNA samples from cells were extracted on days 0, 11 and 14 (or 15) for qRT-PCR analysis.
(4) The analyzed genes are shown in FIGS.

  The differentiation process induced the expression of albumin (day 14/15; FIGS. 12 and 13). In addition, the above operation was performed using naive ADSC as a control, the gene expression level was checked, and compared with hepatocyte-like cells produced from rota-ADSC (FIG. 14). Naive ADSCs showed increased TAT and TDO2 expression, but no albumin-expressing cells were obtained.

1-2. Rota-ADSC toward mesoderm lineage
<Procedure for adipocyte differentiation>
(1) According to the method described in Example 1, rota-ADSC colony (P10) obtained by ADSC (P9) suspension / vibration system was seeded at a concentration of about 70000 cells per well. (3 wells; 100% confluent; 24 well plates). The rota-ADSC was then maintained for 8 days in culture dishes containing the following media: 10% FBS, 100 units / ml penicillin, 100 microgram / ml streptomycin sulfate, 2 mM glutamine (Gln), 100 × Adipogenic supplement (containing hydrocortisone, isobutylmethylxanthine, indomethacin) (Mouse Mesenchymal Stem Cell Functional Identification Kit, R & D Systems, Inc.).
(2) Medium changes are performed every 2-3 days, and RNA samples from cells are extracted on day 3 (naive ADSC), day 0 (rota-ADSC) and day 8 for qRT-PCR analysis did.
(3) Expression transcripts from multiple genes (PPARg2 and Tfap2a) whose expression is restricted to the adipocyte lineage were amplified using qRT-PCR.

  In differentiated cells, Pparg and Tfap2a were expressed 20-30 times higher than the starting rota-ADSC cells or naive ADSCs. The differentiated form after 8 days confirmed the qRT-PCR result (FIGS. 15 and 16).

<Procedure for bone cell differentiation>
(1) In accordance with the method described in Example 1, rota-ADSC colonies (P10) obtained by ADSC (P9) suspension / vibration system were seeded at a concentration of about 25000 cells per well. (50% confluent; 3 wells in a 24-well laminin coated plate). Naive ADSC (P11) (same batch ADSC used for rota-ADSC induction) was used as a control for the same procedure.
(2) rota-ADSC and naive ADSC were maintained for 21 days in laminin-coated dishes containing the following media: 10% FBS, 100 units / ml penicillin, 100 microgram / ml streptomycin sulfate, 2 mM An αMEM medium containing glutamine (Gln) and 20 × mOsteogenic supplement (containing ascorbate-phosphate, β-glycerolphosphate and recombinant BMP-2) (Mouse Mesenchymal Stem Cell Functional Identification Kit, R & D Systems, Inc.).
(3) Medium exchange was performed every 2-3 days and RNA samples were extracted from naive ADSC and rota-ADSC on days 0 and 21 for qRT-PCR analysis.

  RT-PCR confirmed bone cell differentiation. Specifically, all four genes checked for differentiation were upregulated 21 days after treatment (FIG. 17). There was no difference in the differentiation potential of the two populations investigated, naive ADSC and rota-ADSC had the same ability in this particular procedure.

1-3. Rota-ADSC toward the ectoderm lineage
<Procedure for neural differentiation>
(1) According to the method described in Example 1, 5 × 10 ⁵ naive ADSCs (P9) were used for the production of rota-ADSC colonies (P10). All the resulting rota-ADSC colonies (P10) were seeded in 10 cm Petri dishes (1st step) and maintained for 9 days in the following medium: 50x B-27 supplement (cat # 17504044, Life Technologies), 100 Neurobasal medium containing microgram / ml kanamycin, 2 mM glutamine (Gln), 30 ng / ml mouse basic fibroblast growth factor (mbFGF) and 30 ng / ml mouse epidermal growth factor (mEGF).
(2) Cells are passaged into laminin-coated dishes and the medium is α-MEM containing the following medium: 2% FBS, 25 ng / ml mbFGF and 25 ng / ml brain-derived neurotrophic factor (BDNF) The medium was changed (second step). This second step was as long as 10 days.
(3) Each medium was changed every 2-3 days, and RNA samples from cells were extracted on days 0 and 19 for qRT-PCR analysis.

  The medium used was Kuroda et al 2013 (Kuroda, Y., et al., Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat. Protocols, 2013. 8 (7): p. 1391. -1415), but all of the human growth factors used in the Kuroda et al 2013 protocol were changed to mouse growth factors.

  After two-step differentiation, neurogenin-2 was expressed similarly to motor neuron precursors and motor neurons. Gfap expression indicates the formation of glial fibrillary acidic protein normally found in astrocytes. Thus, rota-ADSC differentiated into neuron-like cells (FIG. 18).

Claims (6)

  A method for producing stem cells, comprising a step of culturing stromal cells in the incubator while rotating and / or vibrating the incubator.   The method according to claim 1, wherein the stromal cells are mesenchymal stromal cells.   The method according to claim 2, wherein the mesenchymal stromal cells are adipose tissue-derived stromal cells.   The stem cell manufactured by the method of any one of Claims 1-3.   The manufacturing method of the differentiated cell including the process of culture | cultivating the obtained stem cell in a differentiation-inducing culture medium after the culture | cultivation process of the stromal cell in the method of any one of Claims 1-3.   6. The method of claim 5, wherein the differentiated cells are selected from the group consisting of hepatocytes, adipocytes, bone cells and nerve cells.

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