WO2023106122A1 - 間葉系譜への分化に特化した神経堤細胞の製造方法 - Google Patents

間葉系譜への分化に特化した神経堤細胞の製造方法 Download PDF

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WO2023106122A1
WO2023106122A1 PCT/JP2022/043474 JP2022043474W WO2023106122A1 WO 2023106122 A1 WO2023106122 A1 WO 2023106122A1 JP 2022043474 W JP2022043474 W JP 2022043474W WO 2023106122 A1 WO2023106122 A1 WO 2023106122A1
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mesenchymal
stem cells
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真 池谷
大介 上谷
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Kyoto University NUC
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Definitions

  • the present invention relates to a method for producing neural crest cells specialized for differentiation into the mesenchymal lineage. More specifically, the present invention relates to a method for producing neural crest cells specialized for differentiation into mesenchymal lineage, including a step of culturing pluripotent stem cell-derived neural crest cells under specific conditions.
  • MSC Mesenchymal Stem Cell
  • osteocytes chondrocytes
  • adipocytes a cell population that can differentiate into osteocytes, chondrocytes, and adipocytes in vitro. It is present in several tissues such as tissue, synovium, dental pulp, and umbilical cord blood. Since bone marrow-derived MSCs have long been used for hematopoietic stem cell transplantation, they are considered safe for medical use. Primary MSCs are currently used to treat several clinical complications such as graft-versus-host disease, Crohn's disease, ischemic cardiomyopathy, and stroke. These wide applications demonstrate the importance of MSCs in cell therapy (Non-Patent Document 1).
  • the quality of MSCs fluctuates depending on the donor's health condition and dosage. Furthermore, the number of MSCs obtained from the elderly is usually low, as the production of MSCs in vivo decreases with age. Therefore, it is essential to develop a method for stably supplying high-quality MSCs for cell therapy. Various efforts have been made in this direction (improvement of culture medium, optimization of oxygen conditions, etc.), but a method for stably supplying high-quality MSCs has not yet been established.
  • NCCs neural crest cells
  • MSCs are known to differentiate via mesoderm cells or neural crest cells (NCC) (for example, Non-Patent Document 2).
  • NCCs are multipotent ectodermal cells that develop from intermediates between neuroectoderm and epidermal ectoderm during vertebrate development, and include ectodermal lineage cells such as peripheral neurons and glial cells, and osteocytes via MSCs.
  • mesoderm cells such as chondrocytes and adipocytes
  • endoderm cells such as hepatocytes and pancreatic cells.
  • Non-Patent Literature Non-Patent Literature 3
  • this induction method contains animal-derived components and is not suitable for cell therapy.
  • Patent Document 1 a method for expanding and culturing NCC while maintaining its characteristics. Specifically, neural crest cells induced to differentiate from pluripotent stem cells were cultured in a medium containing a GSK3 ⁇ inhibitor at a concentration exceeding 1 ⁇ M that exhibited an effect equivalent to that of CHIR99021. We succeeded in expanding and culturing neural crest cells that retain their potential. However, Patent Document 1 does not disclose neural crest cells specialized for differentiation into the mesenchymal lineage.
  • the subject of the present invention is a method for obtaining mesenchymal stem cells suitable for cell therapy and suitable for regenerative medicine applications such as bone, cartilage, and muscle without containing heterologous animal-derived components (i.e., xeno-free). And as a preliminary step, it is to provide a method for obtaining xeno-free neural crest cells specialized for differentiation into the mesenchymal lineage from pluripotent stem cells.
  • the present inventors are studying a method for producing mesenchymal stem cells (MSC) from pluripotent stem cells via neural crest cells (NCC), and are improving the method described in Non-Patent Document 3. developed a method for inducing the differentiation of pluripotent stem cells into NCCs under xeno-free conditions, and a method for inducing the differentiation of NCCs into MSCs. However, it was found that the differentiation ability of NCCs induced by the above method changed with passage when cultured for the purpose of maintenance under xeno-free conditions.
  • NCCs which are essentially cells that can differentiate into various cell types such as bone, cartilage, muscle, sensory neurons, glial cells, melanocytes, and adipocytes, were maintained in xeno-free medium containing ALK inhibitors, EGF, and FGF. When cultured, it was found that after a certain passage, the ability to differentiate into neural cells and melanocytes was lost. That is, the present inventors succeeded in producing novel neural crest cells that do not have the ability to differentiate into nervous system cells or melanocytes.
  • MSCs with a high survival rate and differentiation efficiency can be obtained, It was found that the skull and muscles were efficiently repaired when transplanted to the defect site.
  • the present inventors have completed the present invention as a result of further studies based on these findings.
  • the present invention is as follows.
  • a method for producing neural crest cells specialized for differentiation into the mesenchymal lineage from pluripotent stem cells 1) culturing pluripotent stem cells in a culture medium containing an ALK inhibitor and a GSK-3 ⁇ inhibitor under xeno-free and feeder-free conditions to obtain neural crest cells; culturing under xeno-free and feeder-free conditions in a culture medium containing an ALK inhibitor, EGF, and FGF and substantially free of a GSK-3 ⁇ inhibitor;
  • the pluripotent stem cells are derived from humans.
  • a neural crest cell obtained by the method according to any one of [1] to [6].
  • a neural crest cell having the following characteristics (A) to (C).
  • B) derived from pluripotent stem cells (B) expressing one or more genes selected from TWIST, DLX1, and CDH11 C) not expressing PAX3 and/or SOX10
  • the neural crest cell according to [8] further having the following characteristics (D) and/or (E).
  • a method for producing mesenchymal stem cells comprising the step of culturing the neural crest cells of any one of [7] to [9] in a medium for inducing differentiation of mesenchymal stem cells.
  • a mesenchymal stem cell obtained by the method of [10].
  • a method for producing mesenchymal cells comprising the step of culturing the mesenchymal stem cells according to [11] in a mesenchymal cell differentiation-inducing medium.
  • a therapeutic agent for cell transplantation comprising the mesenchymal stem cells of [11] or the mesenchymal cells of [13-1] or [13-2].
  • Treatment of tissue damage or disease comprising administering or transplanting an effective amount of the mesenchymal stem cells according to [11] or the mesenchymal cells according to [13-1] or [13-2] to a subject. Method of treatment.
  • neural crest cells specialized for differentiation into the mesenchymal lineage can be produced under xeno-free conditions, and the neural crest cells serve as starting cells for mesenchymal stem cells that are important for cell transplantation therapy.
  • the neural crest cells serve as starting cells for mesenchymal stem cells that are important for cell transplantation therapy.
  • mesenchymal stem cells can be produced from the neural crest cells under xeno-free conditions. By passing through neural crest cells, the possibility of contamination with undifferentiated iPS cells into mesenchymal stem cells can be reduced.
  • the mesenchymal stem cells obtained by the present invention can be suitably used for the treatment of diseases accompanied by bone, cartilage and muscle damage (cell transplantation therapy).
  • iNCCs Efficient induction of iNCCs from iPSCs under xeno-free conditions.
  • A Schematic of neural crest induction protocol using AK03N and Basic03 xeno-free media.
  • B Colony morphology during induction. Phase-contrast images obtained on days 0, 4, and 10. SOX10 (red) expression was merged at day 10 in the far right panel. Scale bar, 200 ⁇ m.
  • E Expression of marker genes in sorted CD271 high (CD271H) and CD271 low (CD271L) cells.
  • F Cells were stained with anti-TUBB3 antibody (green) and anti-peripherin antibody (red).
  • C Dot plot analysis of NCC induction on day 10. The x-axis shows CD271 expression and the y-axis shows SSEA4 expression.
  • B Mesoderm (MES) and endoderm (END) gene expression in 1231A3 iPSCs, induced NCCs (D2, D4, D6, D8, D10) at days 2-10, and high expression of CD271-positive cells. Heatmap showing (H) and low expressing (L) fractions.
  • C Regional markers of 1231A3 iPSCs, expression of forebrain (FB), midbrain (MB), midbrain-hindbrain boundary (MHB), hindbrain (HB), and spinal cord (SC) genes, days 2-10. Heat map showing the induced NCCs (D2, D4, D6, D8, D10) in 2D and high-expressing (H) and low-expressing (L) fractions of CD271-positive cells.
  • PC1 principal component 1
  • PC2 principal component 2.
  • A Schematic of neural crest expansion culture protocol.
  • B Phase-contrast images of PN0, 2, 4, and 7. Scale bar, 100 ⁇ m.
  • (D) Expression of marker genes during expansion. mRNA expression of each gene was analyzed using RT-qPCR during expansion culture and presented as a relative value using the level of 1231A3h iPSCs as 1.0. Data are mean ⁇ SD, n 3.
  • E Immunostaining for SOX10 (purple), TWIST (green) and DLX1 (red) during expansion culture. Scale bar, 100 ⁇ m.
  • F Differentiation of peripheral neurons from PN1 (upper panel) or PN4 (lower panel) in expanded cultures. Cells were stained with anti-TUBB3 antibody (green) and anti-peripherin antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 200 ⁇ m. Xeno-free induction of iMSCs from expanded iNCCs.
  • A Schematic of mesenchymal stromal cell (MSC) induction protocol using PRIME-XV MSC XSFM xeno-free medium.
  • (C) Cell numbers during MSC induction. Data are mean ⁇ SDn 3.
  • A Phase contrast of XF-iMSCs (PN4), human adipocyte-derived mesenchymal stem cells (hAC-MSCs), human bone marrow-derived mesenchymal stem cells (hBM-MSCs), and human umbilical cord-derived MSCs (hUC-MSCs) image. Scale bar, 200 ⁇ m.
  • C 1231A3 iPSCs, induced NCCs (NCCs) at day 10, pluripotent stem cells (PSCs) of XF-iMSCs, hAC-MSCs, hBM-MSCs, and hUC-MSCs, neural crest cells (NCCs) , and a heatmap showing the expression of mesenchymal stromal cell (MSC) genes.
  • N NCC-induced (NCC), XF-iMSCs, hAC-MSCs, hBM-MSCs, and hUC-MSCs at day 10.
  • A Schematic of XF-iMSC transplantation into the mouse skull.
  • (C) Expression marker genes in GM-cultured aggregates (white) and OIM-cultured aggregates (black). The mRNA expression of each gene was analyzed by RTqPCR in GM- and OIM-cultured aggregates and shown as a relative value using the level of GM-cultured aggregates as 1.0. Data are mean ⁇ SD, n 4. ** P ⁇ 0.01.
  • Black boxes in the left column indicate the same regions as those in the middle column of serial sections.
  • the right column shows high magnification images of the white boxes in the middle column. Scale bars, 500 ⁇ m (left column), 100 ⁇ m (middle column), and 10 ⁇ m (right column).
  • Enhanced skull regeneration with transplanted cell clusters (XF-iMSCs).
  • A CT scan image of the implanted mouse skull. The pierced area is indicated by a red circle.
  • C Lateral image of the implanted skull.
  • Sections were stained with HE or Azan (bottom right).
  • the black box in (C) has the same area as in (D) for serial sections.
  • Scale bar 500 ⁇ m.
  • (D) Lateral fluorescence image of the implanted skull. Sections were stained with anti-human vimentin (green). Nuclei were stained with DAPI (blue). The right column shows higher magnification images of the white boxes in the left column. Scale bars, 100 ⁇ m (left column) and 10 ⁇ m (right column), respectively.
  • TA tibialis anterior
  • (C) Cross-sectional area per cell in the injured field 2 or 5 weeks after transplantation. Data are mean ⁇ SD, n 3. * P ⁇ 0.05. ns: no significant difference.
  • B Immunofluorescence images of neuronal (left) and melanocyte (right) derived cells from PN1 (upper panel) and PN4 (lower panel) of NCC expansion cultures. Left panel: cells were stained with anti-GFAP antibody (green) and anti-peripherin antibody (red). Right panel: cells were stained with anti-MITF antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 50 ⁇ m.
  • Muscle regeneration by transplanted XF-iMSCs Muscle regeneration by transplanted XF-iMSCs.
  • A Cross section of injured TA muscle 5 weeks after transplantation. Sections were stained with laminin (white), MYH4 (red), and MYH4 (red)/human lamin A/C (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 ⁇ m.
  • B Cross section of injured TA muscle 5 weeks after transplantation. Sections were stained with laminin (white), MYH3 (red)/human lamin A/C (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 ⁇ m. Most MYH3-positive cells are found around the transplanted XF-iMSCs.
  • the present invention provides neural crest cells specialized for differentiating from pluripotent stem cells into mesenchymal lineage (can be rephrased as "mesenchymal cell population").
  • a method of manufacturing a cell is provided.
  • such methods include: 1) culturing pluripotent stem cells in a culture medium containing an ALK inhibitor and a GSK-3 ⁇ inhibitor under xeno-free and feeder-free conditions to obtain neural crest cells; culturing under xeno-free and feeder-free conditions in a culture medium containing an ALK inhibitor, EGF, and FGF and substantially free of a GSK-3 ⁇ inhibitor; including (hereinafter sometimes referred to as "the production method of the present invention").
  • Pluripotent stem cells are cells that can differentiate into various tissues and cells with different morphologies and functions in the body, and cells of any lineage of the three germ layers (endoderm, mesoderm, ectoderm). Also refers to stem cells that have the ability to differentiate. Pluripotent stem cells used in the present invention include, for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryos derived from cloned embryos obtained by nuclear transfer. stem cells (nuclear transfer Embryonic stem cells: ntES cells), multipotent germline stem cells (“mGS cells”), embryonic germline stem cells (EG cells), but preferably iPS cells (more preferably human iPS cells).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • mGS cells multipotent germline stem cells
  • EG cells embryonic germline stem cells
  • iPS cells more preferably human iPS cells
  • the pluripotent stem cell is an ES cell or any cell derived from a human embryo
  • the cell may be a cell produced by destroying the embryo or a cell produced without destroying the embryo. Although it may be, preferably the cell is produced without destroying the embryo.
  • ES cells are stem cells with pluripotency and the ability to proliferate through self-renewal, established from the inner cell mass of early mammalian embryos (for example, blastocysts) such as humans and mice.
  • ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and later ES cell lines were established in primates such as humans and monkeys (J.A. Thomson et al. al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. ., 55:254-259; J.A.
  • ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder.
  • ES cells can be established using only a single blastomere of a cleavage-stage embryo prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113- 117), it can also be established using developmentally arrested embryos (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).
  • nt ES cells are cloned embryo-derived ES cells produced by nuclear transfer technology, and have almost the same characteristics as fertilized egg-derived ES cells (Wakayama T. et al. (2001), Science, 292 (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). That is, nt ES (nuclear transfer ES) cells are ES cells established from the inner cell mass of a cloned embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell.
  • nt ES cells For the production of nt ES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (above) is used (Wakayama Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra edition), pp. 47-52).
  • nuclear transfer the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal and cultured for several hours to initialize the egg.
  • ES cell line used in the present invention if it is a mouse ES cell, for example, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (RIKEN), etc. can be used.
  • RIKEN inGenious targeting laboratory
  • RIKEN RIKEN
  • human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, the National Center for Child Health and Development, and Cellartis are available.
  • human ES cell lines include CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains distributed by ESI Bio, H1 strains and H9 strains distributed by WiCell Research.
  • iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors).
  • iPSCs by introducing four factors, Oct3/4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPSCs derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al.
  • Nanog-iPSCs were established by selecting Nanog expression as an indicator (Okita, K., Ichisaka, T., and Yamanaka, S. (2007 ). Nature 448, 313-317.), iPSCs produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) etc. can also be used.
  • induced pluripotent stem cells established by introducing the four factors of OCT3/4, SOX2, NANOG, and LIN28 produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Unexamined Patent Application Publication No. 2008-307007) and the like can also be used.
  • iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used as induced pluripotent stem cell lines.
  • human iPSC strains include HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains of RIKEN; 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, etc., and 1231A3 strain is preferred.
  • mGS cells are testis-derived pluripotent stem cells and are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into cells of various lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be produced (Kanatsu-Shinohara M. et al. 2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004), Cell, 119:1001-1012). It is capable of self-renewal in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and can reproduce by repeating passages under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra edition), pp. 41-46, Yodosha (Tokyo, Japan)).
  • GDNF glial cell line-derived neurotrophic factor
  • EG cells are cells with pluripotency similar to ES cells, which are established from embryonic primordial germ cells. It can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factors (Matsui Y. et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).
  • the species from which the pluripotent stem cells are derived is also not particularly limited. and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees.
  • a preferred species of origin is human.
  • neural crest cells are cells that develop between the neuroectoderm and the epidermal ectoderm when the neural tube is formed from the neural plate in the early stage of development. Cells with multipotency and self-proliferative ability to differentiate into many types of cells such as stem cells, osteocytes, chondrocytes and melanocytes.
  • the term "neural crest cell” means a cell that expresses TFAP2A and one or more genes selected from SOX10, PAX3, NGFR, CDH6, TWIST, DLX1, and CDH11.
  • cells shall include “cell populations” unless otherwise specified.
  • a cell population may be composed of one type of cell, or may be composed of two or more types of cells.
  • ALK inhibitor means a substance having inhibitory activity against receptors belonging to the ALK (activin receptor-like kinase) family.
  • ALK is also called type I TGF ⁇ receptor, and controls cell proliferation, cell differentiation, cell death, and the like through signal transduction mainly through activation of Smad (R-Smad).
  • R-Smad Smad
  • Those that formed the type II TGF ⁇ receptor form a dimer, and the dimer associates with two molecules of the type I TGF ⁇ receptor to form a heterotetramer. Formation of the heterotetramer causes the type II TGF ⁇ receptor to phosphorylate the type I TGF ⁇ receptor, which induces kinase activity of the type I TGF ⁇ receptor and phosphorylates downstream Smads.
  • the "ALK inhibitor” used in the present invention is not particularly limited as long as it can inhibit any stage of the above signal transduction.
  • Substances that inhibit phosphorylation of type I TGF ⁇ receptors substances that inhibit phosphorylation of Smads (eg, Smad2, Smad3, etc.) by phosphorylated type I TGF ⁇ receptors, and the like.
  • ALK-1, ALK-2, ALK-3, ALK-4, ALK-5, ALK-6 and ALK-7 are known as ALK
  • the ALK inhibitors used in the present invention include: In particular, inhibitors against ALK-5 (also referred to as "TGF ⁇ inhibitors”) are preferred.
  • Examples of the ALK inhibitor used in step 1) of the production method of the present invention include SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazole-2 -yl)-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4- (3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), A83-01 (3-(6-methylpyridin-2-yl)-1- phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline),
  • the concentration of the ALK inhibitor in the medium is appropriately adjusted depending on the type of ALK inhibitor to be added, and is typically 1 to 50 ⁇ M, preferably 2 to 40 ⁇ M, more preferably. is 5-20 ⁇ M.
  • the concentration in the medium is Typically, it is 1-40 ⁇ M, preferably 5-20 ⁇ M, more preferably 10 ⁇ M.
  • GSK3 ⁇ inhibitor means a substance having inhibitory activity against GSK3 ⁇ (glycogen synthase kinase 3 ⁇ ).
  • GSK3 (glycogen synthase kinase 3) is a type of serine/threonine protein kinase, and is involved in many signaling pathways involved in glycogen production, apoptosis, and stem cell maintenance. GSK3 has two isoforms, ⁇ and ⁇ .
  • the "GSK3 ⁇ inhibitor” used in the present invention is not particularly limited as long as it has GSK3 ⁇ inhibitory activity, and may be a substance having both GSK3 ⁇ inhibitory activity and GSK3 ⁇ inhibitory activity.
  • Examples of the GSK3 ⁇ inhibitor used in step 1) of the production method of the present invention include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4- (2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H -imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CP21R7 (3-(3-amino-phenyl)-4-(1-methyl-1H-indol-3-yl) -pyrrole-2,5-dione), LY2090314 (3-[9-Fluoro-1,2,3,4-tetrahydro-2-(1-piperidinyl
  • antisense oligonucleotides and siRNA against GSK3 ⁇ mRNA antibodies that bind to GSK3 ⁇ , dominant-negative GSK3 ⁇ mutants, etc. can also be used as GSK3 ⁇ inhibitors, and these are commercially available or according to known methods. Can be synthesized.
  • the concentration of the GSK3 ⁇ inhibitor in the medium is appropriately adjusted depending on the type of GSK3 ⁇ inhibitor added, and is, for example, 0.01-20 ⁇ M, preferably 0.1-10 ⁇ M.
  • the concentration in the medium is typically 0.1-1 ⁇ M, preferably 0.5-1 ⁇ M, more preferably 1 ⁇ M.
  • the culture period in step 1) of the production method of the present invention is not particularly limited as long as it is a period during which the desired cells can be obtained. days (especially 10 days).
  • the culture density of cells is not particularly limited as long as the cells can grow.
  • 1.0 ⁇ 10 1 to 1.0 ⁇ 10 6 cells/cm 2 preferably 1.0 ⁇ 10 2 to 1.0 ⁇ 10 5 cells/cm 2 , more preferably 1.0 ⁇ 10 3 to 1.0 ⁇ 10 4 cells/cm 2 , more preferably 3.0 ⁇ 10 3 to 1.0 ⁇ 10 4 cells/cm 2 (especially 3.6 ⁇ 10 3 cells/cm 2 ).
  • the cells Prior to step 1) of the production method of the present invention, the cells may be cultured in a medium containing neither the TGF ⁇ inhibitor nor the GSK3 ⁇ inhibitor, and the culture period is a period during which the desired number of cells can be obtained. Well not particularly limited. Typically 2-6 days. Such culture is preferably performed under feeder-free and xeno-free conditions.
  • neural crest cells that maintain multipotency are obtained.
  • Such neural crest cells in addition to the above characteristics of neural crest cells (that is, expressing TFAP2A and expressing one or more genes selected from SOX10, PAX3, NGFR, CDH6, TWIST, DLX1, and CDH11), Cells having the property of expressing SOX10, PAX3, NGFR and CDH6 and not expressing TWIST and/or DLX1.
  • neural crest cells that have maintained multipotency are characterized as expressing TFAP2A, SOX10, PAX3, NGFR and CDH6 and not expressing TWIST, DLX1 and CDH11.
  • maintaining multipotency means having the ability to differentiate into nerve cells, glial cells and mesenchymal stem cells, preferably into osteocytes, chondrocytes and melanocytes in addition to these. It means having differentiation ability.
  • the neural crest cells obtained in step 1) of the production method of the present invention may be subjected to step 2) after sorting by FACS (Fluorescence-Activated Cell Sorting) or the like using high CD271 expression as an index.
  • FACS Fluorescence-Activated Cell Sorting
  • ALK inhibitor used in step 2) of the production method of the present invention those described above (that is, those that can be used in step 1) can be used without particular limitation.
  • a preferred ALK inhibitor is at least one selected from the group consisting of SB431542, A83-01, LDN193189, GW788388, SM16, IN-1130, GW6604 and SB505124.
  • a particularly preferred TGF ⁇ inhibitor is SB431542. These may be used in combination of two or more.
  • the concentration of the ALK inhibitor in the medium is appropriately adjusted depending on the type of ALK inhibitor to be added, typically 1 to 50 ⁇ M, preferably 2 to 40 ⁇ M, more preferably. is 5-20 ⁇ M.
  • the concentration in the medium is Typically, it is 1-40 ⁇ M, preferably 5-20 ⁇ M, more preferably 10 ⁇ M.
  • EGF Extracellular Growth Factor
  • concentration of EGF in the medium is not particularly limited, but is typically 5-100 ng/ml, preferably 20-40 ng/ml, more preferably 20 ng/ml.
  • FGF Fibroblast Growth Factors
  • FGF-2 basic fibroblast growth factor
  • the concentration of FGF in the medium is not particularly limited, but is typically 10-200 ng/ml, preferably 20-40 ng/ml, more preferably 20 ng/ml.
  • the term "substantially free of GSK-3 ⁇ inhibitor” means not only that GSK-3 ⁇ is not detected in the culture medium, but also that 10 nM of CHIR99021 has an effect equivalent to that shown by Cultures without higher concentrations of GSK3 ⁇ inhibitors than those indicated are also included.
  • GSK3 ⁇ inhibitory activity can be measured using the gene expression regulation function of GSK3 ⁇ in the Wnt/ ⁇ -catenin pathway (specifically, the phosphorylation function of ⁇ -catenin) as an index.
  • the method established in Example 9 of Patent Document 1 can be used. Details will be described below.
  • GSK3 ⁇ functions in the phosphorylation of ⁇ -catenin in the absence of Wnt-ligands.
  • Phosphorylated ⁇ -catenin is ubiquitinated and degraded in the proteasome, suppressing gene expression downstream of the Wnt- ⁇ -catenin pathway.
  • ⁇ -catenin is translocated into the nucleus without being degraded, and Wnt- ⁇ - Induces gene expression downstream of the catenin pathway.
  • CellSensor LEF/TCF-bla HCT-116 Cell Line (Thermo Fisher, K1676) is integrated to stably express LEF/TCF, and the reporter gene (beta-lactamase reporter gene) is under the control of LEF/TCF. incorporated to be expressed. Reporter gene expression in the absence of Wnt-ligand in this cell line is indicative of inhibition of GSK3 ⁇ function ( ⁇ -catenin phosphorylation function). An assay using the same cell line can measure the GSK3 ⁇ inhibitory activity of a GSK3 ⁇ inhibitor.
  • the assay is performed according to Invitrogen's protocol (CellSensor (registered trademark) LEF/TCF-bla HCT 116 Cell-based Assay Protocol). Specifically, LEF/TCF-bla HCT-116 Cell was added to the assay medium (OPTI-MEM, 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 100 U/mL/100 ⁇ g/mL Pen/Strep). Suspend (312,500 cells/mL). The cell suspension is seeded into each well of the assay plate (10,000 cells/well) and cultured for 16-24 hours. CHIR99021 is added to the wells (concentration 10 nM) and incubated for 5 hours.
  • CellSensor registered trademark
  • LEF/TCF-bla HCT 116 Cell-based Assay Protocol LEF/TCF-bla HCT 116 Cell-based Assay Protocol. Specifically, LEF/TCF-bla HCT-116 Cell was added to the as
  • beta-lactamase substrate solution LiveBLAzer-FRET B/G (CCF4-AM) Substrate Mixture
  • CCF4-AM LiveBLAzer-FRET B/G
  • GSK3 ⁇ inhibitors other than CHIR99021 that is, GSK3 ⁇ inhibitors to be evaluated
  • fluorescence values at each concentration condition (0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, 10000 nM
  • a calibration curve is prepared according to a standard method, it is possible to determine the concentration exhibiting GSK3 ⁇ inhibitory activity equivalent to that exhibited by 10 nM CHIR99021.
  • the fluorescence value is at the background level, it can be evaluated that GSK-3 ⁇ is below the detection sensitivity in the culture medium.
  • the culturing period in step 2) of the production method of the present invention is not particularly limited as long as it is a period during which the desired cells can be obtained. It is days. As shown in the following examples, step 2) of the production method of the present invention can maintain NCCs capable of differentiating into mesenchymal stromal cells for a long period of time (at least 45 days or more). Therefore, the culture period may be 45 days or longer.
  • the culture density of cells is not particularly limited as long as the cells can grow.
  • 1.0 ⁇ 10 1 to 1.0 ⁇ 10 6 cells/cm 2 preferably 1.0 ⁇ 10 2 to 1.0 ⁇ 10 5 cells/cm 2 , more preferably 1.0 ⁇ 10 3 to 1.0 ⁇ 10 5 cells/cm 2 , more preferably 5.0 ⁇ 10 3 to 5.0 ⁇ 10 4 cells/cm 2 (especially 1.0 ⁇ 10 4 cells/cm 2 ).
  • steps 1) and 2) of the production method of the present invention cells are cultured under feeder-free and xeno-free conditions.
  • feeder-free means a medium or culture condition that does not contain other cell types that play a supporting role (i.e., feeder cells) used to condition the cells to be cultured.
  • xeno-free means a medium or culture conditions that do not contain components derived from organisms different from the species of cells to be cultured.
  • the feeder-free and xeno-free medium used in the present invention is not particularly limited, but StemFit (registered trademark) AK02 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03 medium (Ajinomoto Co., Ltd.), StemFit (registered trademark) Basic03 medium , CTS (registered trademark) KnockOut SR XenoFree Medium (Gibco), mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), and the like.
  • AK03 medium is preferred.
  • the medium contains, for example, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercapto It may contain one or more serum replacements such as ethanol, 3'-thiol glycerol, and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics. It may also contain one or more substances such as substances antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine.
  • KSR Knockout Serum Replacement
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • albumin transferrin
  • apotransferrin fatty acids
  • insulin insulin
  • collagen precursors insulin
  • trace elements 2-mercapto
  • 2-mercapto 2-mercapto
  • Cultivation in steps 1) and 2) of the production method of the present invention may be either suspension culture or adherent culture, as long as the desired cells can proliferate, but preferably any culture in steps 1) and 2) Mo is an adherent culture.
  • the term "suspension culture” refers to culture performed under conditions that maintain cells or cell aggregates floating in a culture medium, i.e., between cells or cell aggregates and a culture vessel. means culturing under conditions that do not allow the formation of strong cell-substratum junctions.
  • adherent culture refers to culture under conditions that form strong cell-substrate bonds between cells or aggregates of cells and cultureware or the like.
  • the surface of the culture vessel is artificially treated for the purpose of improving adhesion to cells (e.g., basement membrane preparations, fibronectin, laminin or fragments thereof, entactin, Extracellular matrices such as collagen, gelatin, synthmax, vitronectin, etc., coating treatment with polymers such as polylysine, polyornithine, etc., or surface treatment such as positive charge treatment).
  • cells e.g., basement membrane preparations, fibronectin, laminin or fragments thereof, entactin, Extracellular matrices such as collagen, gelatin, synthmax, vitronectin, etc.
  • coating treatment with polymers such as polylysine, polyornithine, etc.
  • surface treatment such as positive charge treatment
  • laminin or fragments used in the present invention include laminin-111 and its E8 region-containing fragment, laminin-211 and its E8 region-containing fragment (e.g., iMatrix-211), laminin-121 or its E8 region-containing fragment, Laminin-221 or its E8 region-containing fragment, Laminin-332 or its E8 region-containing fragment, Laminin-3A11 or its E8 region-containing fragment, Laminin-411 or its E8 region-containing fragment (e.g. iMatrix-411) , laminin-421 or its E8 region-containing fragment, laminin-511 or its E8 region-containing fragment (e.g.
  • laminin-521 or its E8 region-containing fragment laminin-213 or A fragment containing the E8 region, laminin-423 or a fragment containing the E8 region, laminin-523 or a fragment containing the E8 region, laminin-212/222 or a fragment containing the E8 region, laminin-522 or the E8 region Fragments containing Among them, laminin-511 or a fragment containing its E8 region is preferred.
  • the culture vessel used for floating culture is not particularly limited as long as it allows "suspension culture", and can be appropriately determined by those skilled in the art.
  • Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multidishes, microplates, microwell plates, micropores, multiplates, multiwell plates, chamber slides, petri dishes, tubes, trays, culture bags, or roller bottles.
  • a bioreactor is exemplified as a vessel for suspension culture.
  • These culture vessels are preferably cell non-adhesive in order to enable suspension culture.
  • the non-cell-adhesive culture vessel the surface of the culture vessel is not artificially treated (eg, coated with an extracellular matrix or the like) for the purpose of improving adhesion to cells.
  • the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, culture is performed in an atmosphere containing CO 2 , and the CO 2 concentration is preferably about 2 to 5%. .
  • neural crest cells specialized for differentiation into the mesenchymal lineage are obtained. Accordingly, in another aspect of the present invention, neural crest cells obtained by the production method of the present invention are also provided. Such neural crest cells typically have the characteristics of neural crest cells (i.e., express TFAP2A and express one or more genes selected from SOX10, PAX3, NGFR, CDH6, TWIST, DLX1, and CDH11). ), and expresses one or more genes selected from TWIST, DLX1, and CDH11, and does not express PAX3 and/or SOX10. In one embodiment, neural crest cells that are specialized for mesenchymal lineage differentiation are characterized as expressing TFAP2A, TWIST, DLX1 and CDH11 and not expressing PAX3 and SOX10.
  • neural crest cells having all of the following properties (A) to (C) are provided.
  • the neural crest cells of the present invention can also be read as cells having all of the following properties (A') to (C').
  • A' derived from pluripotent stem cells (B') expressing TFAP2A and expressing one or more genes (preferably all three genes) selected from TWIST, DLX1, and CDH11 (C ') does not express PAX3 and/or SOX10 (preferably PAX3 and SOX10)
  • Neural crest cells obtained by the production method of the present invention or neural crest cells of the present invention may have all of the following properties (I) to (III).
  • the neural crest cells obtained by the production method of the present invention or the neural crest cells of the present invention induced mesenchymal stem cells as compared to neural crest cells not subjected to step 2) of the production method of the present invention.
  • the viability and differentiation efficiency of cases can be increased (eg, 2-fold or more, 4-fold or more, 10-fold or more).
  • the term “gene expression is enhanced” means that the expression level of the gene is higher than in the cells before step 2) of the production method of the present invention (e.g., 2-fold, 3-fold) means twice, four times, five times or more).
  • the expression of the gene is reduced or eliminated means that the expression level of the gene is lower than that of the cells before performing step 2) of the production method of the present invention (e.g., 1/2-fold, 1 /5-fold, 1/10-fold, or less) or no expression.
  • the term “specialized in differentiation to the mesenchymal lineage” means having no (lost) ability to differentiate into neural cells and melanocytes, and mesenchymal stem cells and the cells It means having (maintaining) the ability to differentiate into osteocytes, chondrocytes and adipocytes via
  • a cell specialized for differentiation into mesenchymal lineage is defined as "a cell with a tendency to easily differentiate into mesenchymal lineage.” lineage)” can also be read.
  • the neural crest cells obtained by the production method of the present invention or the neural crest cells of the present invention further exhibit any one (preferably all) of the following properties (D) to (F): have.
  • the term "not capable of differentiating into neural cells” means that the cells were treated with a neural differentiation induction medium (N2 supplement, B27 supplement, 2 mM L-glutamine, 10 ng/mL BDNF, 10 ng/mL Does not differentiate into TuBB3-positive and Peripherin-positive peripheral neurons or GFAP-positive glial cells after 3 weeks of culture in Neurobasal TM medium containing GDNF, 10 ng/mL NT-3 and 10 ng/mL NGF means that Therefore, even if cells are differentiated into peripheral neurons or glia under culture conditions other than those described above, they "do not have the ability to differentiate into nervous system cells.”
  • “does not have the ability to differentiate into melanocytes” means that cells are melanocyte differentiation induction medium (medium StemFit (registered trademark) Basic03 containing 1 ⁇ M CHIR99021, 25 ng/mL BMP4, 100 nM Endothelin-3) It means that they do
  • the terms "expressing” or “positive” a gene are used to mean at least "production of mRNA encoded by the gene” unless otherwise specified. is used in the sense of including "the production of a protein encoded by”. Therefore, when the production of mRNA encoded by the gene is detected at least by the following method (quantitative RT-PCR), it can be said that the gene is expressed. On the other hand, if the production of mRNA encoded by the gene is not detected (i.e., below the detection limit) by the following method (quantitative RT-PCR), or if it is at background levels, the gene is not expressed or is negative. It can be said.
  • mRNA when not detected by the following method, even if mRNA production is detected by a method other than the following method, the gene is not expressed in the present specification.
  • mRNA also includes pre-mRNA. A method for detecting gene expression will be described in detail below.
  • Total RNA is purified using RNeasy Mini Kit (Qiagen) and treated with DNase-one Kit (Qiagen) to remove genomic DNA. Reverse transcribe 500 ng of total RNA to obtain single-stranded cDNA using PrimeScript RT Master Mix (Takara) according to the manufacturer's instructions. Quantitative PCR using Thunderbird SYBR qPCR Mix (Toyobo) is performed using QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems).
  • the neural crest cells obtained by the production method of the present invention or the neural crest cells of the present invention have the ability to differentiate into mesenchymal stem cells. Therefore, in another aspect, a method for producing mesenchymal stem cells (hereinafter referred to as , sometimes referred to as “method for producing mesenchymal stem cells of the present invention”), and mesenchymal stem cells obtained by the method are provided.
  • mesenchymal stem cell means a cell that has self-renewal ability and at least differentiation ability into osteocytes, chondrocytes and adipocytes. In the method for producing mesenchymal stem cells of the present invention, it is preferable to perform all steps under feeder-free and xeno-free conditions.
  • the mesenchymal stem cell differentiation-inducing medium is not particularly limited as long as it can induce differentiation of the neural crest cells obtained by the production method of the present invention or the neural crest cells of the present invention into mesenchymal stem cells by culturing.
  • a mesenchymal stem cell differentiation-inducing medium a basal medium containing bovine serum (eg, ⁇ MEM medium) may be used, but a commercially available xeno-free medium for mesenchymal stem cell proliferation is preferred.
  • xeno-free media for mesenchymal stem cell proliferation include, for example, PRIME-XV MSC Expansion XSFM (Fujifilm Wako Pure Chemical Industries, Ltd.), Cellartis (registered trademark) MSC Xeno-Free Culture Medium (Takara Bio Inc.), MSC NutriStem (registered trademark) XF medium (Sartorius Stedim Japan K.K.) and the like.
  • the culture period in the method for producing mesenchymal stem cells of the present invention is not particularly limited as long as the desired cells can be obtained, but it is typically 5 to 50 days, preferably 10 to 30 days, more preferably 14 days. is.
  • FACS analysis for expression of cell surface antigens eg, CD73, CD44, CD45 and CD105
  • the culture density of cells is not particularly limited as long as the cells can grow.
  • 1.0 ⁇ 10 1 to 1.0 ⁇ 10 6 cells/cm 2 preferably 1.0 ⁇ 10 2 to 1.0 ⁇ 10 5 cells/cm 2 , more preferably 1.0 ⁇ 10 3 to 1.0 ⁇ 10 5 cells/cm 2 , more preferably 5.0 ⁇ 10 3 to 5.0 ⁇ 10 4 cells/cm 2 (especially 1.0 ⁇ 10 4 cells/cm 2 ).
  • mesenchymal stem cells differentiated cells
  • mesenchymal stem cells comprising the step of culturing mesenchymal stem cells obtained by the method for producing mesenchymal stem cells of the present invention, in a mesenchymal cell differentiation-inducing medium.
  • a production method hereinafter sometimes referred to as a “method for producing mesenchymal cells of the present invention”
  • mesenchymal cells obtained by the method are provided. Examples of such mesenchymal cells include osteocytes, chondrocytes, and adipocytes.
  • the osteocyte differentiation induction medium a basal medium containing 10% FBS, 0.1 ⁇ M dexa-methasone, 50 ⁇ g/ml ascorbic acid and 10 mM ⁇ -glycerophosphate (e.g. ⁇ MEM) or a commercially available xeno-free osteocyte differentiation induction medium (e.g. , MSCgo TM Rapid Osteogenic Differentiation Medium (Biological Industries)) and the like.
  • the method of differentiating into osteocytes includes, for example, seeding 4 ⁇ 10 4 mesenchymal stem cells on a gelatin-coated 12-well plate and culturing them in the osteocyte differentiation-inducing medium for 30 days. mentioned. Calcified nodules may be detected by alizanin red staining to confirm differentiation into osteocytes.
  • Chondrocyte Differentiation Induction Medium contains 1% (v/v) ITS+premix, 0.17 mM AA2P, 0.35 mM proline, 0.1 mM dexamethasone, 0.15% (v/v) glucose, 1 mM sodium pyruvate, 2 mM GlutaMAX, Basal medium (e.g., DMEM/F12) containing 40 ng/ml PDGF-BB, 100 ng/ml TGF- ⁇ 3, 10 ng/ml BMP4, and 1% (v/v) FBS or commercially available xeno-free chondrocytes differentiation-inducing media (eg, MSCgo TM Chondrogenic XF (Biological Industries)); Specifically, the method for differentiation into chondrocytes is, for example, spotting 5 ⁇ l of the mesenchymal stem cell suspension on a fibronectin-coated plate, culturing for 1 hour, and adding 1 ml of the above-menti
  • Adipocyte differentiation induction medium includes basal medium containing 60 ⁇ M indomethacin, 0.5 mM IBMX, 0.5 ⁇ M hydrocortisone, and commercially available xeno-free adipocyte differentiation induction medium (e.g., hMSC - Human Mesenchymal Stem Cell Adipogenic Differentiation Medium (Lonza), MSCgo TM Adipogenic XF (Biological Industries)) and the like.
  • Specific examples of adipocyte differentiation methods include a method in which 4 ⁇ 10 4 mesenchymal stem cells are seeded on a gelatin-coated plate and cultured in the osteocyte differentiation-inducing medium for 32 days. Adipocyte differentiation may be confirmed by Oil Red O staining.
  • mesenchymal stem cells obtained by the method for producing mesenchymal stem cells of the present invention and mesenchymal cells (differentiated cells) obtained by the method for producing mesenchymal cells of the present invention (hereinafter referred to as these may be collectively referred to as "the mesenchymal cells of the present invention"), when transplanted to bone, cartilage and muscle defect sites, may have superior repair ability than cells obtained by conventional methods. . Therefore, the mesenchymal cells of the present invention can be suitably used for cell transplantation therapy.
  • a therapeutic agent for cell transplantation comprising the mesenchymal cells of the present invention (hereinafter sometimes referred to as "the therapeutic agent for cell transplantation of the present invention”).
  • the therapeutic agent for cell transplantation of the present invention an effective amount of the mesenchymal cells of the present invention is administered or transplanted to a mammal to be treated (e.g., human, mouse, rat, monkey, bovine, horse, pig, dog, etc.), tissue (e.g., , bone tissue, cartilage tissue, adipose tissue, muscle tissue (eg, skeletal muscle tissue, etc.), and methods for treating damage (including defects) or diseases are also included in the present invention.
  • tissue injury treatment also includes regeneration of injured tissue.
  • the purpose of the transplantation of the mesenchymal cells of the present invention into a living body may be the direct regeneration of damaged tissues, or the indirect effect of the factors secreted by the mesenchymal cells of the present invention (e.g., paracrine effect, etc.).
  • mesenchymal stem cells can exert therapeutic effects in patients with acute myocardial infarction, stroke, multiple system atrophy (MSA), graft-versus-host disease, Crohn's disease, ischemic cardiomyopathy, spinal cord injury, and the like.
  • the term "substantially identical" means that the HLA genotype matches the transplanted cells to the extent that an immunosuppressive agent can suppress an immune reaction.
  • HLA-DR 3 loci or 4 loci including HLA-C are somatic cells that have the same HLA type. If sufficient cells cannot be obtained due to age or physical constitution, it is possible to embed the cells in a capsule such as polyethylene glycol or silicone, or a porous container to avoid rejection. be.
  • the mesenchymal cells of the present invention are produced as parenteral preparations such as injections, suspensions, infusions, etc. by mixing with pharmaceutically acceptable carriers according to conventional methods. Therefore, in one aspect, there is also provided a method for producing a cell transplantation therapy comprising the step of formulating the mesenchymal cells of the present invention.
  • a production method may include a step of preparing the mesenchymal cells of the present invention.
  • a step of preserving the mesenchymal cells of the present invention can also be included.
  • Pharmaceutically acceptable carriers that can be included in the parenteral formulation include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). of aqueous liquids for injection.
  • Cell transplantation therapeutic agents of the present invention include, for example, buffers (e.g., phosphate buffers, sodium acetate buffers), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, and the like.
  • the cells can be suspended in the above aqueous solution at about 1 ⁇ 10 6 to about 1 ⁇ 10 8 cells/mL. good.
  • the dosage or the amount of transplantation of the mesenchymal cells or the pharmaceutical composition of the present invention and the frequency of administration or the frequency of transplantation can be appropriately determined depending on the age, body weight, symptoms, etc. of the mammal to be administered.
  • the cell transplantation therapeutic agent of the present invention is provided in a cryopreserved state under the conditions normally used for cryopreservation of cells, and can be thawed before use.
  • it may further contain serum or its substitute, an organic solvent (eg, DMSO), and the like.
  • the concentration of serum or its substitutes is not particularly limited, but can be about 1 to about 30% (v/v), preferably about 5 to about 20% (v/v).
  • the concentration of the organic solvent is not particularly limited, but can be from 0 to about 50% (v/v), preferably from about 5 to about 20% (v/v).
  • Human iPSCs (1231A3, 1381A5, 1381B5, 1383D2, and 1383D10) were cultured in StemFit AK03N (Ajinomoto, Tokyo, Japan) on cell culture plates or dishes coated with iMatrix-511 (Nippi, Tokyo, Japan), as previously described. Japan). Medium was changed every 2 days.
  • Human mesenchymal stem/stromal cells hAC-MSCs, hBM-MSCs, hUC-MSCs
  • PromoCell Heidelberg, Germany.
  • MSCs were cultured in PRIME-XV MSC Expansion XSFM medium (Fujifilm Irvine Scientific, Tokyo, Japan) on fibronectin (Millipore, Bedford, CA, USA)-coated culture dishes. Medium was changed every 3 days.
  • Human dermal fibroblasts (HDFs) were obtained from CellApplications (San Diego, CA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific) (Thermo Fisher Scientific, Waltham, USA). Massachusetts, USA). Medium was changed every 3 days.
  • DMEM Dulbecco's Modified Eagle Medium
  • NCC neural crest cells
  • Human iPS cells were seeded in StemFit AK03N medium in StemFit iMatrix-511 coated plates or dishes at a density of 3.6 ⁇ 10 3 cells/cm 2 and maintained in culture for 4 days.
  • NCC induction cells were treated with 10 ⁇ M SB431542 (Fujifilm Wako) and 1 ⁇ M CHIR99021 (Axon Medchem, Reston, VA) in StemFit Basic03 (equivalent to AK03N without bFGF, Ajinomoto, Tokyo, Japan). USA) for 10 days.
  • Cell numbers were counted using a Countess II FL (Thermo Fisher Scientific). The medium was changed every 2 days from day 0 to 6 and every day from day 7 to 10.
  • NCC differentiation For differentiation of peripheral neurons and glial cells, 1 ⁇ 10 5 CD271-high-expressing sorted NCCs were seeded on fibronectin-coated 12-well plates and supplemented with B27 supplement (Thermo Fisher Scientific), N-2. supplement (Thermo Fisher Scientific), 2 mM L-glutamine (Fujifilm Wako Pure Chemical, Tokyo, Japan), 10 ng/mL BDNF (Fujifilm Wako Pure Chemical), GDNF (Fujifilm Wako Pure Chemical), NT- 3 (Fuji Film Wako Pure Chemical) and Neurobasal (Thermo Fisher Scientific) medium supplemented with NGF (Fuji Film Wako Pure Chemical) for 3 weeks. Medium was changed every 3 days. Differentiation was confirmed by immunostaining with peripherin, TUBB3, and GFAP.
  • CD271 high-expressing sorted NCCs were seeded on fibronectin-coated 12-well plates and treated with 1 ⁇ M CHIR99021, 25 ng/ml BMP4 (R&D Systems, Minneapolis, MN, USA), and Cultured in Basic03 supplemented with 100 nM Endothelin-3 (Tocris, Bristol, UK) for 10 days. Medium was changed every 2 days. Differentiation was confirmed by immunostaining with MITF.
  • NCC expansion culture CD271-high expressing sorted NCC were seeded in Basic03 supplemented with 10 ⁇ M SB431542, 20 ng/ml EGF, and FGF2 on fibronectin-coated plates at a density of 1 ⁇ 10 4 cells/cm 2 . Medium was changed every 3 days. For passaging, cells were dispersed with Accutase (Innovative Cell Technologies, San Diego, Calif., USA) and replated onto fibronectin-coated plates at a density of 1 ⁇ 10 4 cells/cm 2 .
  • NCC NCC 5 ⁇ 10 5 NCC were suspended in 500 ⁇ l of STEM-CELLBANKER GMP grade (Takara, Kusatsu, Japan) and frozen using CoolCell Cell Freezing Containers (Bioscience, Kyoto, Japan). frozen.
  • NCCs (Induction of mesenchymal stromal cells (XF-iMSCs) from NCC) Expanded NCCs (passage 4; PN4) were seeded in Basic03 supplemented with 10 ⁇ M SB431542, 20 ng/ml EGF, and FGF2 on fibronectin-coated plates at a density of 1 ⁇ 10 4 cells/cm 2 . The next day, the medium was replaced with PRIME-XV MSC Expansion XSFM medium. Cell morphology began to change approximately 4 days after induction. Passages were performed every 4 days with Accutase at a density of 1 ⁇ 10 4 cells/cm 2 . Human MSC markers (CD44, CD73, CD90 and CD105) were analyzed by FACS 14 days after hMSC induction.
  • PN4 mesenchymal stromal cells
  • XF-iMSCs For chondrogenic differentiation, 1.5 ⁇ 10 5 XF-iMSCs were mixed in 5 ⁇ l chondrogenic medium (DMEM/F12, Thermo Fisher Scientific), 1% (v/v) ITS+ premix (Corning, Corning, NY, USA). USA), 0.17 mM AA2P (Sigma, St.
  • induced cells were fixed with 4% paraformaldehyde (PFA) (Fujifilm Wako) for 30 min and rinsed with phosphate-buffered saline (PBS). Next, these cells were stained with an alcian blue solution (1% alcian blue (Muto Kagaku Co., Ltd., Tokyo, Japan)) at 25° C. for 1 hour.
  • PFA paraformaldehyde
  • alcian blue solution 1% alcian blue (Muto Kagaku Co., Ltd., Tokyo, Japan)
  • 4 ⁇ 10 4 XF-iMSCs were seeded in 0.1% gelatin-coated 12-well plates and cultured in MSCgo Rapid Osteogenic Differentiation Medium (Biological Industries, Cromwell, CT, USA) for 30 days. Medium was changed every 3 days.
  • Oil Red O staining Differentiation properties were confirmed by Oil Red O staining. Cells were fixed with 10% formalin at room temperature for 1 hour and then incubated with 0.3% Oil Red O staining solution (Sigma) for 20 minutes. Non-specific staining was removed by washing several times with water.
  • FACS sorting FACS was performed using an AriaII instrument (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. The antibodies used are shown in Table 1. In all experiments, isotype controls were used to eliminate non-specific background signals.
  • RNA was purified using RNeasy Micro Kit (Qiagen) and treated with DNase-one kit (Qiagen) to remove genomic DNA. 10 ng of total RNA was reverse transcribed to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA library synthesis of the Ion AmpliSeq transcriptome was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and the Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer's protocol. . Barcode-labeled cDNA libraries were analyzed using the Ion S5 XL System (Thermo Fisher Scientific) and the Ion 540 Chip Kit (Thermo Fisher Scientific).
  • XF-C-iMSCs were prepared as previously reported (Stem Cell Res Ther 8, 101 (2017)) with minor modifications. Briefly, XF-iMSCs were seeded onto fibronectin-coated 48-well plates (Corning, Corning, NY, USA) at a density of 1.0 ⁇ 105 cells/well and plated with Prime-XV MSC Expansion XSFM for 4 days. cultured. To obtain XF-C-iMSCs, confluent cells formed on a cell sheet consisting of ECM produced by the MSCs themselves were scratched using a micropipette tip and then detached.
  • the sheet-like iMSC/ECM complexes detached from the bottom of the plate were transferred to a 24-well ultra-low binding plate (Corning) and rolled up to form round cell clusters.
  • Cell clumps were maintained in Prime-XV MSC Expansion XSFM or MSCgo Osteogenic differentiation medium (Biological Industries) for 2, 5, or 10 days.
  • XF-C-iMSCs were fixed with 4% PFA in PBS. Samples were embedded in paraffin and serially sectioned at 5 ⁇ m thickness. Samples were then stained with hematoxylin and eosin (H&E) or alizarin red S and viewed using a Nikon Eclipse E600 microscope (Nikon, Kawasaki, Japan).
  • H&E hematoxylin and eosin
  • alizarin red S viewed using a Nikon Eclipse E600 microscope (Nikon, Kawasaki, Japan).
  • XF-C-iMSCs cultured in MSCgo Osteogenic Differentiation medium for 2 days were transplanted into the defect without an artificial scaffold.
  • a transplant of XF-C-BMMSCs maintained in the same medium was also used as a control.
  • the skin incision was then sutured using 4-0 silk sutures.
  • Micro CT analysis Mice were sacrificed 28 days after surgery and cranial regions were imaged using SkyScan1176 in vivo micro-CT (Bruker, Billerica, MA, USA). Three-dimensional reconstructions were generated using CTVOL software (Bruker). The volume of newly formed bone within the bone defect was determined using CT-An software (Bruker).
  • slides were incubated for 10 minutes in staining solution (mixture of equal volumes of 10% potassium dichromate and 10% trichloroacetic acid) and washed with H2O .
  • staining solution mixture of equal volumes of 10% potassium dichromate and 10% trichloroacetic acid
  • H2O washed with H2O .
  • the slides were then incubated in an azocarmine G solution (0.1% azocarmine G, 1% acetic acid) for 30 minutes.
  • Specimens were briefly washed with H 2 O, identified with aniline alcohol (0.1 mL of aniline dissolved in 100 mL of 95% ethanol), washed with acetic alcohol and H 2 O, and phosphotungstic acid (5%).
  • mice For XF-iMSC transplantation, 8- to 16-week-old NSG mice were purchased from Charles River Japan (Yokohama, Japan). Mice were anesthetized with 3% Forane inhalant liquid (AbbVie, North Chicago, IL, USA). The middle portion of the tibialis anterior (TA) muscle was then continuously crushed by direct clamping with forceps for 1 min under constant pressure determined using the same manometer (Biores Open Access 2, 295-306 (2013)).
  • TA tibialis anterior
  • XF-iMSCs or human dermal fibroblasts were suspended in ⁇ MEM (2 ⁇ 10 5 cells/50 ⁇ l) and injected into the center of the tibialis anterior muscle injury site 24 hours after injury using a 27G microsyringe. bottom.
  • mice were sacrificed at 3 days, 2 weeks, and 5 weeks after injury.
  • the tibialis anterior muscle was placed on tragacanth gum (Fujifilm Wako Pure Chemical Industries) and frozen with liquid nitrogen (PLoS One 8, e61540 (2013)).
  • Serial sections (10 ⁇ m) were cut using a cryostat. Sections were stained with H&E and viewed using an Olympus BX51 microscope (Olympus, Tokyo, Japan). Four sections prepared from the middle of each TA muscle sample were stained and the entire cross section of all sections was photographed and analyzed. The highest value among the values obtained from the 4 thin slices taken was adopted as the data for each sample.
  • Immunofluorescence images were acquired using a Zeiss LSM 710 laser scanning confocal microscope (Zeiss). Area measurements and cell counts were performed using hybrid cell counting software BZH3C (Keyence).
  • BZH3C hybrid cell counting software
  • FIG. 9C images stained with anti-laminin antibody were analyzed with Keyence image analysis software to calculate the average area of muscle fibers.
  • FIG. 9E images stained with anti-MYH4 antibody were analyzed with Keyence image analysis software and the area of positive areas was calculated.
  • FIG. 9G images stained with anti-MYH3 antibody were counted for the number of positive fibers.
  • Myoblast differentiation from mouse neonatal myoblasts Myoblasts were isolated from neonatal C57BL/6 mice (Clea Japan, Tokyo) as previously described (Stem Cell Res 30, 122-129 (2016)). Myoblasts were treated with 20% FCS, 10% horse serum, 0.5% chicken embryo extract, 2.5 ng/ml FGF2, 10 ⁇ g/ml gentamicin, 1% antibiotic-antimitotic, and They were cultured in high-glucose DMEM (Proliferation Medium: PM) containing 2.5 ⁇ g/ml plasmocin prophylaxis reagent.
  • RNA-seq data supporting the results of the examples of the present application are registered in the Gene Expression Omnibus (GEO) database under accession codes GSE206048, GSE206128, and GSE206172.
  • the proteome data supporting the findings of the examples of the present application are registered in the jPOSTrepo (Japan Proteome Standard Repository) database under the accession code JPST001693.
  • Example 1 Induction of NCCs from Human iPSCs under Xeno- free Conditions Previous induction protocols were modified to induce NCCs from human iPSCs (hiPSCs) under xeno-free conditions. In the original protocol, STO cell lines transformed with neomycin resistance and the LIF gene (SNL) were transformed into stromal feeders in growth factor-depleted Matrigel-coated culture dishes extracted from Engelbreth-Holm-Swarm mice. A cell-maintained iPSC line was used.
  • hiPSCs were cultured for 4 days until colonies were formed before initiating NCC induction.
  • the original protocol used bovine serum albumin (BSA) during NCC induction.
  • BSA bovine serum albumin
  • the basal medium of the BSA-containing medium was replaced with Stemfit Basic03.
  • the medium corresponds to AK03N minus bFGF.
  • the induction efficiency was analyzed using fluorescence-activated cell sorting (FACS) using the CD271 antibody, or the antibody and the SSEA4 antibody.
  • FACS data revealed that almost no SSEA4-positive undifferentiated iPS cells were detected ( ⁇ 0.05%), and the ratio of CD271 high-positive cells reached a peak of 90% (Fig. 2B). , C).
  • the robustness of this protocol was demonstrated in 1231A3 and other feeder-free and xeno-free iPSC lines (1231A3, 1381A5, 1381B5, 1383D2, and 1383D10; 71.8 ⁇ 18.3%, 59.0 ⁇ 13.7%, 55.2 ⁇ 17.5%, 15.0 ⁇ 11.1%, respectively). %, and 50.3 ⁇ 13.3%) (Fig. 2D).
  • CD271 high-positive cells appeared by day 4 and gradually increased during induction (Fig. 1D). Since CD271 recognizes the cell surface protein NGFR (p75), cell sorting using CD271 antibody allowed enrichment of NCCs (Fig. 2E). Enrichment was confirmed by higher expression of NCC markers (NGFR, SOX10, TFAP2A and RHOB) in CD271-high cells compared to CD271-low cells (Fig. 1E). We also used quantitative real-time polymerase chain reaction (RT-qPCR) to confirm the expression of NCC and neuronal markers PAX3 and PAX6, and the pluripotent cell marker POUF5F1. It was found that the CD271 low-positive cells contained nerve cells (Fig. 1E).
  • RT-qPCR quantitative real-time polymerase chain reaction
  • TUBB3, peripherin, and GFAP peripheral nervous system cells
  • MITF melanocytes
  • CD271-high expressing cells were TUBB3 positive, peripherin, GFAP and MITF negative (Fig. 10A).
  • TUBB3-positive neurons were induced from CD271 weakly-positive cells, but peripherin-positive peripheral neurons were hardly detected, and MITF-positive pigment cells were not detected at all. This result was consistent with the finding that CD271 weakly positive cells contained neuroectoderm (Fig. 10B).
  • NCC neuroectoderm
  • ectodermal markers including neuroectoderm (PAX6 and DACH1), neural plate border (PAX3, PAX7, ZIC1, MSX2, and TFAP2A), and NCC (SOX10, FOXD3, NGFR, ITGA4, and SNAI2) markers was up-regulated. Consistent with the RT-qPCR data, CD271-high expressing cells highly expressed NCC markers, whereas CD271-low expressing cells highly expressed neuroectodermal markers.
  • PCA principal component analysis
  • Example 2 Verification of Neuronal Differentiation Potency of Xeno-free NCCs Expanded with TGF ⁇ Inhibitor SB431542, EGF, and bFGF (hereinafter referred to as "SB"), EGF, and bFGF, which can be grown in a chemically defined medium (including BSA) (PLoS One 9, e112291 (2014), Exp Cell Res 316, 1148- 1158 (2010)).
  • BSA chemically defined medium
  • TFAP2A a pan-NCC marker
  • RHOB another NCC marker
  • NCCs acquired mesenchymal characteristics.
  • the differentiation properties of expanded NCCs were assessed by culturing NCCs under neuronal, glial, melanocyte and MSC inducing conditions.
  • Example 3 Differentiation of NCCs Expanded under Xeno-free into MSCs
  • NCCs were cultured in serum-containing medium or xeno-free MSC medium (PRIME-XV® MSC Expansion XSFM) (FIG. 5A)
  • These cells also displayed a morphology (Fig. 12A) and gene expression profile (Fig. 12B,C) consistent with MSCs.
  • NCC-derived xeno-free induced MSCs (NCC-derived xeno-free induced MSCs; hereinafter referred to as "XF-iMSCs") under chondrogenesis-, osteogenesis-, and adipogenesis-inducing conditions, cartilage and bone, respectively. , which may differentiate into adipose tissue (Fig. 5E-G).
  • XF-iMSCs NCC-derived xeno-free induced MSCs
  • Example 4 Comparison of XF-iMSCs and tissue-derived MSCs MSCs isolated from different tissues or prepared by different methods have different characteristics. Therefore, we compared XF-iMSCs with different types of MSCs.
  • Figure 6A transcriptome analysis of XF-iMSCs and tissue-derived primary human MSCs (bone marrow-derived MSCs (hBM-MSCs), adipose-derived MSCs (hAC-MSCs), and umbilical cord-derived MSCs (hUC-MSCs).
  • Example 5 Contribution of XF-iMSCs to skull regeneration As XF-iMSCs differentiated into osteocytes under xeno-free conditions using MSC Go Osteogenic XF® (Fig. 5F), the contribution of XF-iMSCs to tissue regeneration We decided to investigate the contribution of iMSC.
  • XF-iMSCs were cultured in 48-well tissue culture plates containing xeno-free MSC medium for 4 days and manually detached from the wells as a sheet to prevent clump (XF-Clump-iMSC, hereinafter “XF-C-iMSC”) formation.
  • GM xeno-free MSC medium
  • OFIM osteocyte-inducing medium
  • XF-C-iMSCs day 5 XF-C-iMSCs (OIMs) were isolated from the skull of immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Implanted into a 1.6 mm hole.
  • XF-C-BMMSC osteogenesis-induced hBM-MSC cell aggregates
  • Figs. 7E and F, 15 were used as a positive control.
  • Example 6 Enhanced Skeletal Muscle Regeneration by XF-iMSC Transplantation
  • XF-iMSCs were transplanted into the tibialis anterior muscle of immunodeficient NOD/SCID/IL2Rgamma null (NSG) mice.
  • NSG immunodeficient NOD/SCID/IL2Rgamma null mice.
  • TA a muscle crush model
  • XF-iMSCs and human dermal fibroblasts (HDFs) were transplanted into TA-injured mice.
  • anti-MYH4 mature muscle fiber marker
  • anti-MYH3 embryonic and fetal muscle fiber marker, also expressed in regenerating muscle fibers
  • anti-laminin antibody base membrane marker
  • immunohistochemistry A chemical analysis was performed. At 5 weeks, long diameter myofibers stained with MYH4, consistent with MYH4, a marker of mature skeletal muscle (Fig. 16A). No staining was detected with anti-MYH3 antibody, which is consistent with MYH3, a marker expressed during the early stages of muscle regeneration (Fig. 16B).
  • MYH3-positive cells increased in the XF-iMSC transplanted group compared with the control group (Fig. 9F and G).
  • the number of MYH3-positive cells was higher in the XF-iMSC transplanted group than in the control group.
  • the number of MYH3-positive cells was lower in the XF-iMSC transplanted group than in the control group after 2 weeks.
  • Example 7 Acceleration of Myotube Differentiation by In Vitro XF-iMSC Conditioned Medium - iMSC conditioned medium was injected. A slight increase in the number of MYH3-positive cells was observed ( Figure 18), but there was no significant difference between unconditioned and conditioned media.
  • neural crest cells specialized for differentiation into the mesenchymal lineage can be produced, and such neural crest cells are useful as starting cells for mesenchymal stem cells and mesenchymal cells.
  • the mesenchymal stem cells and mesenchymal cells obtained by the present invention not only directly regenerate damaged tissues, but also exhibit indirect effects due to the factors secreted by the cells. Useful in regenerative medicine.

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