WO2018199142A1 - Method for producing neural crest cells and sympathetic neurons - Google Patents

Abstract

Pluripotent stem cells are cultured in a culture solution containing FGF2, retinoic acid and BMP4 to thereby produce neural crest cells. The neural crest cells are cultured in a culture solution containing a cytokine and BMP4 to thereby produce sympathetic neuron precursors. Further, the sympathetic neuron precursors are cultured in a culture solution containing a neurotrophic factor to thereby produce sympathetic neurons.

Description

Method for producing neural crest cells and sympathetic neurons

The present invention relates to a method for producing neural crest cells from pluripotent stem cells, and a method for producing sympathetic progenitor cells and further sympathetic neurons from neural crest cells.

Sympathetic nerves are peripheral autonomic nerves that control involuntary functions such as circulation, breathing, digestion, sweating / temperature regulation, endocrine function, reproductive function, and metabolism. Developmentally, sympathetic neurons are known to be derived from trunk neural crest cells, and various factors involved in differentiation induction have been identified in animal models. Although there have been some reports on methods for inducing sympathetic neurons from pluripotent stem cells, differentiation efficiency is not sufficient (Non-Patent Documents 1 to 4).

Huang, M. et al. Generating trunk neural crest from human pluripotent stem cells. Sci. Rep. 6, 19727 (2016). Oh, Y. et al. Functional coupling with cardiac muscle promotes maturation of hPSC-derived sympathetic neurons. Cell Stem Cell 19, 95-106 (2016). Zeltner, N. et al. Capturing the biology of disease severity in a PSC based model of familial dysautonomia. Nat. Med. 22, 1421-1427 (2016). Denham, M. et al. Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system. Stem Cells 33, 1759-1770 (2015).

As described above, there are some reports on the sympathetic differentiation protocol, but the differentiation efficiency is not sufficient. The lack of specific cell surface markers and the lack of reports on methods for inducing differentiation of trunk neural crest cells seem to hinder the development of differentiation-inducing systems from human pluripotent stem cells to sympathetic nerves. It is done.
Accordingly, an object of the present invention is to efficiently produce sympathetic neurons from pluripotent stem cells.

The inventors first developed an efficient protocol for obtaining functional sympathetic neurons via neural crest cells (NCC) by examining each differentiation stage in detail using PHOX2B-eGFP reporter-expressing pluripotent stem cells Established. The present inventors have found that sympathetic neurons can be obtained from pluripotent stem cells with high purity by this protocol, and the present invention has been completed.

That is, the present invention provides the following inventions.
[1] A method for producing neural crest cells, comprising culturing pluripotent stem cells in a culture medium containing FGF (Fibroblast growth factor) 2, retinoic acid and BMP (Bone morphogenetic protein) 4.
[2] The concentration of FGF2 is 1 ng / ml to 100 ng / ml, the concentration of retinoic acid is 10 nM to 10 μM, and the concentration of BMP4 is 5 ng / ml to 150 ng / ml. A method for producing neural crest cells.
[3] Pluripotent stem cells are cultured in a culture solution containing GSK (Glycogen synthase kinase) 3β inhibitor and TGF (Transforming growth factor) β inhibitor, and then in a culture solution containing FGF2, retinoic acid and BMP4. The method for producing neural crest cells according to [1] or [2], which is cultured.
[4] The method for producing a neural crest cell according to any one of [1] to [3], wherein the culture is performed by suspension culture.
[5] The method for producing neural crest cells according to any one of [1] to [4], comprising a selection step using CD49d.
[6] The method for producing a neural crest cell according to any one of [1] to [5], wherein the pluripotent stem cell is an induced pluripotent stem cell.
[7] A method for producing sympathetic progenitor cells, comprising culturing neural crest cells in a culture solution containing cytokine and BMP4 to induce sympathetic progenitor cells.
[8] The method for producing sympathetic progenitor cells according to [7], wherein the cytokines are EGF (Epidermal Growth Factor) and FGF2.
[9] The concentration of BMP4 is 5 ng / ml to 150 ng / ml, the concentration of EGF is 1 ng / ml to 100 ng / ml, and the concentration of FGF2 is 1 ng / ml to 100 ng / ml. [8] A method for producing a sympathetic progenitor cell according to claim 1.
[10] The method for producing sympathetic progenitor cells according to any one of [7] to [9], wherein the culture is performed in suspension culture.
[11] The method for producing sympathetic progenitor cells according to any one of [7] to [10], wherein the neural crest cells are obtained by the method according to any one of [1] to [6].
[12] A neural crest cell is produced by the method according to any one of [1] to [6], and the obtained neural crest cell is used to sympathize with the method according to any one of [7] to [10]. A method for producing sympathetic neural progenitor cells, which produces neural progenitor cells.
[13] A step of producing sympathetic progenitor cells by the method according to any one of [7] to [12], and sympathy by culturing the obtained sympathetic progenitor cells in a culture solution containing neurotrophic factor A method for producing a sympathetic nerve cell, comprising a step of maturing a neural progenitor cell into a sympathetic nerve cell.
[14] The method according to [13], wherein the neurotrophic factor is NGF (Nerve growth factor), BDNF (Brain-derived neurotrophic factor) and GDNF (Glial cell line-derived neurotrophic factor).
[15] A sympathetic nerve cell obtained by the method according to [13] or [14].
[16] A composition for treating sympathetic neuropathy comprising sympathetic nerve cells obtained by the method according to [13] or [14].

According to the present invention, human sympathetic nerve cells, which have conventionally been difficult to collect, can be produced from human pluripotent stem cells and used for many studies. In recent years, a wide variety of neural cells have been induced to differentiate from human pluripotent stem cells and used for regenerative medicine, creation of disease models, drug discovery screening, neurotoxicity evaluation, and the like. Similarly to the above, sympathetic nerve cells induced to differentiate in the present invention are considered to be applicable to regenerative medicine, creation of disease models, drug discovery, and the like.

The figure which shows the analysis result about the differentiation conditions, cell profile, etc. of PHOX2B :: eGFP introduction | transduction human pluripotent stem cell (hPSC). (A) Diagram of culture conditions for adjusting determination of hPSC anteroposterior and dorsoventral axes. (B) Heat map image showing the percentage of eGFP ⁺ cells on day 10 of differentiation of KhES1 PHOX2B :: eGFP strain differentiated under various conditions. (C) Representative FCM plots of KhES1 PHOX2B :: eGFP strain-derived aggregates on day 10 under conditions (i)-(iv) of b. (D) RT-PCR analysis of aggregates on day 10 under conditions (i)-(iv) for PHOX2B, SOX1, PAX6, SOX10, FOXD3, HOXB1, HOXB2, HOXB4, HOXB6, HOXB8 and HOXC9. The right figure shows the expression pattern of the HOX gene in the lombomere (r1-8) and spinal cord (cervical and thoracic) regions. SB = SB431542, CHIR = CHIR99021, RA = retinoic acid, Pur = purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord. The figure which shows the analysis result about the differentiation conditions, marker expression profile, etc. of PHOX2B :: eGFP introduction | transduction human pluripotent stem cell (hPSC). (A) Diagram of culture conditions for sympathetic nervous system NCC differentiation. (B) Representative images of day 10 aggregates from KhES1 control and KhES1 PHOX2B :: eGFP strain (scale bar = 100 μm). (C) Immunocytochemical analysis of unselected and CD49d ⁺ eGFP ⁺ sorted day 10 differentiated cells for PHOX2B and SOX10 (scale bar = 50 μm). A white line box indicates an enlarged area in the right panel. White arrowheads indicate PHOX2B ⁺ SOX10 ⁻ cells. Determination of PHOX2B ⁺ cells and SOX10 ⁺ cells in (d) c (mean ± SEM, n = 3). (E) Immunocytochemical analysis for HOXB7 on scale 0 and 10 days (CD49d ⁺ eGFP ⁺ sorted) (scale bar = 50 μm). (F) Quantification of HOXB7 ⁺ cells in e (mean ± SEM, n = 3). SB = SB431542, CHIR = CHIR99021, RA = retinoic acid. The figure which shows the analysis result about the differentiation from NCC to a sympathetic nerve cell. (A) Diagram of culture conditions for sympathetic NCC culture after FACS purification. (B) Representative image of neurosphere on the third day after sorting under EF and EFB conditions (scale bar = 100 μm). (C) Quantification by FCM analysis of eGFP ⁺ cells in neurospheres 7 days after sorting under EF and EFB conditions (mean ± SEM, n = 3, P <0.01). (D) Changes in cell number, percentage of eGFP ⁺ cells and percentage of SOX10 ⁺ cells in 28 days of culture under EFB conditions after sorting (mean ± SEM, n = 3). The cell number is described as the rate of change relative to the number on day 0. (E) Diagram of culture conditions for sympathetic nerve cell differentiation after FACS purification. (F) Immunocytochemical analysis for eGFP, PHOX2B, TH, PRPH and DBH on 29th day after selection (scale bar = 50 μm). (G) Quantification of PRPH ⁺ cells, TH ⁺ cells and DBH ⁺ cells in f (mean ± SEM, n = 3). (H) Quantification of noradrenaline released from cultured neurons on the 30th day after selection (mean ± SEM, n = 3). EF = EGF + FGF2, EFB = EGF + FGF2 + BMP4. The figure which shows the analysis result about the induction | guidance | derivation of the high purity sympathetic nerve cell which does not perform cell selection. (A) Diagram of culture conditions for sympathetic nerve cell differentiation without cell sorting. (B) FCM analysis of eGFP ⁺ cells on day 10, 17 and 31 of differentiation shown in a. (C) Quantification of eGFP ⁺ cells in b. Data from three independent experiments are shown in each graph. (D) Immunocytochemical analysis for eGFP, TH, PRPH and DBH on day 32 of differentiation (scale bar = 50 μm). (E) Quantification of PRPH ⁺ cells, TH ⁺ cells and DBH ⁺ cells in d (mean ± SEM, n = 3). (F) Immunocytochemical analysis for PHOX2B and SOX10 on days 10 and 17 and for TH, DBH and PRPH on day 32 (scale bar = 50 μm). The white line box in the image on the 10th day indicates the enlarged area on the left side of the same panel. (G) PHOX2B in day 10 and day 17 of f ⁺ cells and SOX10 ⁺ quantification of cells (mean ± SEM, n = 3). (H) Quantification of PHOX2B ⁺ cells, TH ⁺ cells, DBH ⁺ cells and PRPH ⁺ cells on day 32 of f (mean ± SEM, n = 3). SB = SB431542, CHIR = CHIR99021, RA = retinoic acid. The figure which shows the result about construction | assembly of PHOX2B :: eGFP knock-in hPSC clone. (A) Schematic diagram of PHOX2B gene targeting using TALEN-mediated genome editing. (B) Genomic PCR showing targeted integration at the 3′UTR region of PHOX2B. (C) Quantification of eGFP ⁺ PHOX2B ⁺ cells in cells (PHOX2B / eGFP) Quantitative and PHOX2B ⁺ eGFP ⁺ cells in cells (eGFP / PHOX2B). The left image shows the immunocytochemistry findings for eGFP and PHOX2B at day 10 under CHIR ^{2.0 μM} BMP4 RA ^{100 nM} conditions (scale bar = 50 μm). The right graph shows quantitative data (mean ± SEM, n = 3). DSB = double strand break, CHIR = CHIR99021, RA = retinoic acid. The figure which shows the result about the induction | guidance | derivation of NMP-like cells and their derivatives from hPSC. (A) RT-PCR analysis for SOX2, BRACKYURY, TBX6, HOXB1, HOXB2, HOXB4 and HOXB6 in day 3 aggregates using various concentrations of CHIR99021. (B) Heatmap image showing the percentage of eGFP ⁺ cells on day 10 of differentiation using the 409B2 PHOX2B :: eGFP strain under various conditions. (C) Quantification by FCM analysis of eGFP ⁺ cells and CD49d ⁺ cells on day 10 under CHIR ^{1.5 μM} Pur RA ^{1000 nM} conditions (mean ± SEM, n = 3). (D) Immunocytochemical analysis for eGFP, PHOX2B, ChAT and TUBBIII in neurons derived from eGFP ⁺ CD49d ⁻ cells (day 28, scale bar = 50 μm). (E) Quantification of eGFP ⁺ and ChAT ⁺ cells in d (mean ± SEM, n = 3). CHIR = CHIR99021, RA = retinoic acid, Pur = purmorphamine. The figure which shows the effect of RA in the induction | guidance | derivation of PHOX2B expression NCC. (A) Representative FCM plot of aggregates from day 10 KhES1 PHOX2B :: eGFP strain under CHIR ^{2.0 μM} BMP RA ^{0 nM} and CHIR ^{2.0 μM} BMP RA ^{100 nM} conditions. (B) ^{Immunocytochemical} analysis for PHOX2B and SOX10 on day 10 of differentiation under CHIR ^{2.0 μM} BMP4 RA ^{0 nM} and CHIR ^{2.0 μM} BMP4 RA ^{100 nM} conditions (scale bar = 50 μm). (C) Quantification of SOX10 ⁺ cells and PHOX2B ⁺ cells in b (mean ± SEM, n = 3). CHIR = CHIR99021, BMP = BMP4, RA = retinoic acid. The figure which shows the analysis result of the cell in the aggregate of CHIR ^{2.0micromol BMP4RA 100nM} conditions. (A) Time course FCM analysis of eGFP expression and CD49 expression. (B) Quantification of GFP ⁺ cells and CD49d ⁺ cells in the time course analysis of a (mean ± SEM, n = 3). (C) FCM analysis of eGFP, CD49d and TUBBIII in day 10 aggregates. Intracellular eGFP and TUBBIII were stained after extracellular staining, fixation, and permeabilization of CD49d. (D) Quantification of CD49d ⁺ cells, faint TUBBIII cells, TUBBIII ⁺ cells in FCM analysis of c (mean ± SEM, n = 3). (E) Immunocytochemical analysis for PHOX2B and SOX10 of unselected and CD49d ⁺ eGFP ⁺ sorted day 10 differentiated cells (409B2 PHOX2B :: eGFP strain, scale bar = 50 μm). A white line box indicates an enlarged area in the right panel. White arrowheads indicate PHOX2B ⁺ SOX10 ⁻ cells. (F) Immunocytochemical analysis for HOXB7 on day 10 of differentiation (CD49d ⁺ eGFP ⁺ sorted) (409B2 PHOX2B :: eGFP strain, scale bar = 50 μm). The figure which shows the result about the induction | guidance | derivation of the sympathetic nerve cell by the neurosphere culture | cultivation from CD49d ⁺ eGFP ⁺ cell. (A) FCM analysis (gray) of eGFP expression in neurosphere cells 7 days after sorting with or without BMP4. A parent clone without the PHOX2B :: eGFP reporter was used as a negative control (white). (B) Immunocytochemical analysis for SOX10 on day 7, 14 and 28 after selection. (C) FCM analysis of TUBBIII expression in neurosphere cells at day 7, 14, 21, and 28 after selection (grey). An isotype control was used to quantify the negative population (white). (D) Quantification of TUBBIII ⁺ cells in FCM analysis of c (mean ± SEM, n = 3). (E) Immunocytochemical analysis for eGFP and SMA of cells 14 days after selection that are adherently cultured using BMP4. (F) Neurosphere cell morphology with or without NF treatment. (G) Immunocytochemical analysis of neurons derived from 409B2 PHOX2B :: eGFP hiPSC strain for eGFP, TH, PRPH and DBH (29 days after selection, scale bar = 50 μm). EF = EGF + FGF2, EFB = EGF + FGF2 + BMP4, SMA = smooth muscle actin. The figure which shows results, such as a neurosphere culture procedure and marker expression analysis. (A) Diagram of culture conditions for neuronal cell induction in neurosphere culture. (B) Immunocytochemical analysis for eGFP, TH, PRPH and DBH under various neuronal induction conditions of a (scale bar = 50 μm). (C) Quantification of PRPH ⁺ cells, TH ⁺ cells and DBH ⁺ cells in b (mean ± SEM, n = 3). (D) Diagram of neurosphere cell storage and culture after thawing. (E) Cell number of neurosphere cells 7 days after thawing described as fold change with respect to the number on the thawing day (mean ± SEM, n = 3). (F) Immunocytochemical analysis for eGFP, TH, PRPH and DBH after neuronal cell-induced culture of stored neurosphere cells (scale bar = 50 μm). (G) Quantification of PRPH ⁺ cells, TH ⁺ cells and DBH ⁺ cells in f (mean ± SEM, n = 3). EFB = EGF + FGF2 + BMP4, NFs = neurotrophic factor; NGF, BDNF and GDNF.

The present invention provides a method for producing sympathetic neurons from pluripotent stem cells. The production method includes (1) a step of inducing neural crest cells from pluripotent stem cells,
(2) Inducing sympathetic progenitor cells from neural crest cells, and (3) Inducing sympathetic neurons from sympathetic progenitor cells.
This will be described below.

Pluripotent stem cell A pluripotent stem cell is a stem cell having pluripotency that can be differentiated into many cells existing in a living body and also having proliferative ability, and at least hematopoiesis used in the present invention. Any cell that is derived from a progenitor cell is included. Pluripotent stem cells are preferably derived from mammals, and more preferably derived from humans.
Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic Examples include germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts, umbilical cord blood-derived pluripotent stem cells, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like. A preferred pluripotent stem cell is an iPS cell, more preferably a human iPS cell, from the viewpoint that it can be obtained without destroying an embryo, an egg or the like in the production process.

IPS cell production methods are known in the art and can be produced by introducing reprogramming factors into any somatic cells. Here, the reprogramming factor is, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. genes or gene products are exemplified, and these reprogramming factors may be used alone or in combination. Also good. As combinations of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010/111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008 ), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467- 2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008 ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135 , Feng B, et al. (2009), Nat. Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotechnol., 27: 459-461, Lyssiotis CA, et al. ( 2009), Proc Natl Acad Sci U S A. 106: 8912-8917, Kim89JB, 17et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5 : 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. ( 2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9.

Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines. Specifically, somatic cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) blood cells (peripheral) Blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells) Etc.), differentiated cells such as brain cells, lung cells, kidney cells and fat cells. The mammalian individual from which somatic cells are collected is not particularly limited, but is preferably a human.

Neural crest is a process of inducing neural crest cells from pluripotent stem cells. It is a transient tissue that appears at the boundary between neuroectodermal and epidermal germ layers during neural tube formation in vertebrate development. A group of cells that migrates after deepithelialization is called neural crest cells (J Cell Biochem 107, 1046-52 (2009)).
The neural crest-derived cells are defined by the presence of neural crest markers such as SOX10 and FOXD3, for example.

The step of inducing neural crest cells from pluripotent stem cells preferably includes the following steps.
(I) culturing pluripotent stem cells in a culture medium containing a TGFβ inhibitor and a GSK3β inhibitor;
(Ii) A step of culturing the obtained cells in a medium containing FGF2, retinoic acid and BMP4.

The culture medium used for the culture of pluripotent stem cells for the production of neural crest cells is not particularly limited, but the medium used for animal cell culture is transferred to the basal medium as a TGFβ inhibitor, GSK3β inhibitor or FGF2, retinoic acid and It can be prepared by adding BMP4 or the like. Basal media include, for example, Iscove's'Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F1 ', , Essential 培 地 6 medium, Neurobasal テ ク ノ ロ ジ ー Medium (Life Technologies) and mixed media thereof. Serum may be contained in the medium, or serum-free may be used. If necessary, the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiolglycerol, ROCK inhibitor, Purmorphamine, lipids, amino acids, L-glutamine, non-essential amino acids, It may also contain one or more substances such as vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, heparin and the like.

The TGFβ inhibitor used in step (i) is a small molecule inhibitor that interferes with TGFβ family signaling, such as SB431542, SB202190 (RKLindemanndeet al.,. Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories) and the like, for example, when the TGFβ inhibitor is SB431542, the concentration in the medium is 0.5 μM to 100 μM, more preferably 5 μM to 30 μM.

The GSK3β inhibitor used in step (i) is defined as a substance that inhibits the kinase activity of GSK (glycogen synthase kinase) -3β protein (for example, the ability to phosphorylate β-catenin). Already known. Specific examples thereof include indirubin derivatives such as BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin-3′-oxime), SB216763 (3- (2,4-dichlorophenyl) -4- (1 Maleimide derivatives such as -methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), α-bromomethyl ketone compounds such as GSK-3β inhibitor VII フ ェ (4-dibromo-acetophenone), CHIR99021 ( 6-[(2-{[4- (2,4-dichlorophenyl) -5- (4-methylimidazol-2-yl) pyrimidin-2-yl] amino} ethyl) amino] pyridine-3-carbonitrile) ( WO1999 / 65897; CAS number 252917-06-9), cell membrane permeable phosphorylated peptides such as L803-mts, and derivatives thereof. For example, when the GSK3β inhibitor is CHIR99021, the concentration in the medium is preferably 0.5 μM to 100 μM, more preferably 1 μM to 10 μM.

The concentration of retinoic acid in the medium used in step (ii) is usually 10 nM to 10 μM, preferably 50 nM to 5 μM.

The concentration of BMP4 (Bone morphogenetic protein 4) in the medium used in step (ii) is usually 5 ng / ml to 150 ng / ml, preferably 10 ng / ml to 100 ng / ml, More preferably, it is 20 ng / ml to 80 ng / ml.

The concentration of FGF2 (fibroblast growth factor 2: aka bFGF) in the medium used in step (ii) is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml. ~ 30 ng / ml.

In the production of neural crest cells, the culture method of pluripotent stem cells may be adhesion culture or suspension culture, but suspension culture is preferred. For example, pluripotent stem cells can be subjected to suspension culture after separating colonies cultured until they are 80% confluent with respect to the used dish and dissociating them into single cells. Examples of methods for separating pluripotent stem cells include, for example, a method for separating mechanically, a separation solution having protease activity and collagenase activity (eg, Accutase ™ and Accumax ™), or a separation solution having only collagenase activity. The separation method using is mentioned.
Suspension culture means culturing cells in a non-adherent state in a culture vessel. Although not particularly limited, artificial treatment (for example, coating treatment with an extracellular matrix or the like) to improve adhesion to cells. ) Culture vessels that have not been treated, or treatment that artificially suppresses adhesion (for example, coating treatment with polyhydroxyethylmethacrylic acid (poly-HEMA) or nonionic surfactant polyol (Pluronic F-127, etc.)) Can be carried out using the cultured vessel. In suspension culture, embryoid bodies (EB) are preferably formed and cultured.

In the present invention, the temperature conditions for culturing for producing neural crest cells are not particularly limited, but for example, about 37 ° C. to about 42 ° C., about 37 ° C. to about 39 ° C. are preferable. Further, the culture period is not particularly limited as long as neural crest cells can be obtained. For example, the culture period in step (i) is 1 to 4 days, and the culture period in step (ii) is, for example, 3 to 10 Day.

In the present invention, the obtained neural crest cells are preferably purified and used in the next step. For purification (selection), for example, CD49d (integrin α4) can be used, and the purification method can be a method well known to those skilled in the art, for example, by flow cytometry using an anti-CD49d antibody. The method of refine | purifying using the method of refinement | purification etc. is mentioned.

Step of inducing sympathetic progenitor cells from neural crest cells In the present invention, sympathetic progenitor cells mean cells that can be differentiated into sympathetic nerves by adding neurotrophic factor and culturing, for example, PHOX2B (Nature 399, 366-370 (1999)) and CD49d can be recognized as positive.

Sympathetic neural progenitor cells can be obtained by culturing neural crest cells in a culture medium containing cytokines and BMP4.

The culture medium used for the production of sympathetic progenitor cells in the present invention is not particularly limited, but can be prepared by using a medium used for animal cell culture as a basal medium and adding cytokine and BMP4 thereto.
As the basal medium, the same medium as described above can be used.

The concentration of BMP4 is, for example, 5 ng / ml to 150 ng / ml, preferably 10 ng / ml to 100 ng / ml, more preferably 20 ng / ml to 80 ng / ml.

As cytokines, EGF (Epidermal Growth Factor) and FGF2 are preferred.
The concentration of EGF is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml to 30 ng / ml.
The concentration of FGF2 is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml to 30 ng / ml.

In the production of sympathetic progenitor cells, it is preferable to perform suspension culture using a non-cell-adhesive incubator. As described above, pluripotent stem cells are cultured by forming embryoid bodies (EBs), After obtaining the dyke cells, it is preferable to dissociate the obtained EBs and culture them in a culture solution to which cytokines and BMP4 have been added to form neurospheres and culture.

The culture temperature conditions for culturing neural crest cells to produce sympathetic progenitor cells are not particularly limited, but for example, about 37 ° C. to about 42 ° C., preferably about 37 ° C. to about 39 ° C. are preferable. The culture period is not particularly limited as long as sympathetic progenitor cells can be obtained.For example, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, or 24 More than 30 days, less than 30 days, or less than 48 days.

Process for inducing sympathetic neurons from sympathetic progenitor cells The sympathetic neurons are identified as, for example, positive cells of tyrosine hydroxylase (TH) and / or dopamine β-hydroxylase (DBH), preferably more positive for peripherin (PRPH) It is.

In the present invention, the sympathetic nerve cell can be produced by a method including a step of culturing the sympathetic neural progenitor cell in a culture solution to which a neurotrophic factor is added.
Here, neurotrophic factors are ligands to membrane receptors that play an important role in motor neuron survival and function maintenance, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin. 3 (NT-3), Neurotrophin 4/5 (NT-4 / 5), Neurotrophin 6 (NT-6), Glia cell line-derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF) and LIF . Preferred neurotrophic factors are those selected from the group consisting of NGF, BDNF and GDNF.

The concentration of the neurotrophic factor to be added may be appropriately selected by those skilled in the art depending on its efficacy, and is, for example, 1 ng / ml to 100 ng / ml, and preferably 5 ng / ml to 50 ng / ml.

The culture solution used for the production of sympathetic nerve cells is not particularly limited, but can be prepared by adding a neurotrophic factor to a basal medium as a medium used for culturing animal cells. As the basal medium, a medium as described above can be used.

In the present invention, the temperature conditions for culturing sympathetic progenitor cells for producing neurotrophic factors are not particularly limited, but for example, about 37 ° C. to about 42 ° C., preferably about 37 ° C. to about 39 ° C. are preferable. . The culture period is not particularly limited as long as sympathetic neurons can be obtained.For example, for example, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, or 24 days or more, 30 days or less, or 48 days or less.

<Use for screening sympathetic disorder>
The sympathetic nerve cells obtained by the method of the present invention can also be used for screening for compounds for treating sympathetic neuropathy (for example, pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides). For example, a candidate pharmaceutical compound can be evaluated by adding a sympathetic nerve cell and changing the morphology or function of the cell. As an example of a functional change, it can be evaluated by measuring the amount of norepinephrine produced from the cell. Here, the artificial neural progenitor cell from which the sympathetic nerve cell is derived is preferably a cell exhibiting a phenotype similar to that of the sympathetic neuropathy to be treated, and particularly preferably prepared from a somatic cell affected by the sympathetic neuropathy. It is an induced pluripotent stem cell.

<Application to regenerative medicine>
The sympathetic nerve cell obtained by the method of the present invention can be effectively used in the field of regenerative medicine for normalizing damaged sympathetic nervous system tissue. Therefore, this cell can be a therapeutic cell for diseases associated with disorders of all sympathetic nervous system cells (such as sympathetic nerve damage and autonomic dysfunction).

The present invention will be described more specifically in the following examples, but the scope of the present invention is not limited to these examples.

Method Research Approval The use of human ESC was approved by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan. A research plan for recombinant DNA research was approved by the recombinant DNA experimental safety committee of Kyoto University.

Cell lines KhES1 and KhES3 hESC lines were provided by Dr. Hirofumi Suemori (Research Institute for Regenerative Medicine, Kyoto University). Human iPS cell lines 409B2 and 604A1 were provided by Dr. Shinya Yamanaka (iPS Cell Research Institute, Kyoto University). These cell lines were maintained on Growth Factor Reduced Matrigel matrix (Corning) coated cell culture plates containing mTeSR1 medium (STEMCELL Technologies).

Plasmid construction TALEN Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/) is used to construct a transcriptional activator-like effector nuclease (TALEN) plasmid. A group (RVD) was designed. The TALEN encoding plasmid was assembled using Golden Gate TALEN and TAL Effector Kit 2.0 and its protocol for assembly of the TALEN encoding plasmid (Addgene). A mammalian expression vector containing the modified FokI was received from Dr. Takashi Yamamoto of Hiroshima University.
A 1 kbp PCR amplified homology arm was cloned 3 ′ to the loxP-neo-loxP cassette vector for the construction of the targeting vector. Next, using an In-Fusion HD cloning kit (Clontech), a 1 kbp 5 ′ homology arm (PCR amplified product), a T2A peptide sequence (annealed oligonucleotide pair) and an eGFP open reading frame (first ORF without 1ATG; PCR amplification product) was cloned seamlessly 5 ′ of the loxP-neo-loxP cassette vector. PrimeSTAR GXL DNA polymerase (TaKaRa) was used for PCR amplification, and after cloning all PCR amplified DNA fragments, their sequences were completely sequenced.
For construction of the Cre expression vector, the ORF of Cre (NLS-Cre) containing a nuclear translocation signal was cloned into the multicloning site of the pLV-EF1a-MCS-IRES-RFP-Puro vector (BIOSETTIA).

Transfection and generation of stable strains For genome editing by TALEN, transfection was performed using the superelectroporator NEPA21 (Neppagene) according to the manufacturer's instructions. Specifically, the cells were dissociated into individual cells using StemPro Accutase Cell Dissociation Reagent (Gibco). Next, one million cells in one cuvette were transfected with 2 μg of each TANEN plasmid and 6 μg of targeting vector plasmid, and growth factor containing mTeSR1 medium supplemented with 50 μM Y27632 (Merck Millipore). Reduced Matrigel matrix-coated 6 cm cell culture dishes (BD Falcon) were immediately replated. Cell survival was promoted using Y27632 (10 μM) 24-48 hours after transfection. Selection using 100 mg / ml G418 (Wako Pure Chemical Industries, Ltd.) was started 3 days after transfection to select cells. Surviving clones were isolated 10-14 days after drug selection and expanded for subsequent experiments.
For deletion of Cre-loxP, hPSCs were passaged as usual in a 3.5 cm dish (BD Falcon). Two days later, 3 μg of the Cre expression plasmid was transfected using FuGene HD transfection reagent (Promega) according to the manufacturer's instructions. Selection using 500 ng / ml puromycin (InvioGen) was started 2 days after transfection to select cells. 4 days after transfection, the living cells were dissociated, and mitosis containing primate ES cell culture medium (Reprocell Co., Ltd.) supplemented with 5 ng / mL FGF2 (Wako Pure Chemical Industries, Ltd.) and 10 μM Y27632. Those surviving cells were passaged onto inactivated SNL feeder cells. The next day, Y27632 was removed. Individual colonies were isolated 14 days after passage and grown for subsequent experiments.

Genomic PCR
Genomic DNA was extracted using QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer's instructions. Genomic PCR was performed to detect integration into the genome at the target site using PrimSTAR GXL DNA polymerase.

Differentiation of hPSCs Dissociated hPSCs that had been maintained using the StemProactase cell dissociation reagent for NMP-like cell induction into individual cells, and 96-well ultra-low attachment multi-wall plates (Corning) were used. The cells were immediately reaggregated in 100 μL Essential 6 medium (Gibco) supplemented with 10 μM SB431542 (Sigma Aldrich), various concentrations of CHIR99021 (Merck Millipore) and 10 μM Y27632. 10,000 cells / well). On day 1, 50 μL of Essential 6 medium supplemented with 10 μM SB431542 (Sigma Aldrich) and CHIR99021 at the same concentration as day 0 was added to each well of these 96-well plates.
Day 3 aggregates treated with 1.5 μM, 2.0 μM, or 3.0 μM CHIR99021 for modification of the dorsoventral axis were treated with 20 ng / mL FGF2 and / or retinoic acid (RA, all-trans, The cells were cultured in Essential 6 medium supplemented with (Sigma-Aldrich). BMP4 (50 ng / mL; R & D Systems) or 1 μM Purphamine (Tocris Bioscience) was added to the indicated conditions. The medium was changed every other day until day 10.
Aggregates from day 3 treated with 1.5 μM CHIR99021 for cranial motor neuron differentiation up to day 10 in Essential 6 medium supplemented with 20 ng / mL FGF2, 1 μM RA and 1 μM Purmorphamine. Cultured. The medium was changed every other day. On day 10, the cells were dissociated into individual cells using the StemProactase cell dissociation reagent and eGFP ⁺ CD49d ⁻ cells were selected by fluorescence activated cell sorting (FACS, see below). 1 × Glutamax I (Gibco), N2 and B27 supplements (Gibco), 100 nM Compound E (Abcam), 10 ng / mL BDNF (R & D Systems), 10 ng / mL GDNF (R & D Systems) were added. Sorted cells were cultured on growth factor-reduced Matrigel matrix-coated culture plates containing Neurobasal medium (Gibco) and 20 μM Y27632 was added during the first 2 days after sorting. The medium was changed every other day and the cells were passaged once at 7-10 days after selection.
20 ng / mL FGF2, 100 nM RA and 50 ng / mL BMP4 were added to day 3 aggregates treated with 2.0 μM CHIR99021 for differentiation of sympathetic NCC and SN (Sympathetic neurons) The cells were cultured in Essential 6 medium until day 10. The medium was changed every other day. On day 10, the cell aggregates were dissociated into individual cells using the StemProactase cell dissociation reagent and eGFP ⁺ CD49d ⁺ cells were sorted by FACS. 1 × Glutamax I (Gibco), N2 and B27 supplements, 20 ng / mL FGF2, 20 ng / mL EGF (R & D) in an ultra-low attachment dish (10 cm) or multi-wall plate (6 wells) (Corning) Systems), sorted cells in Neurobasal medium supplemented with 50 ng / mL BMP4 and 2 μg / mL heparin (Sigma Aldrich) were cultured at a density of 500,000 cells / mL. The medium is changed every 3-4 days, and 0.05% trypsin and 10 μg / mL DNaseI (STEMCELL Technologies) are used, followed by gentle pipetting to dissociate the cells and remove the cell mass every 7 days. Passage treatment.
Neurobasal medium supplemented with 1 × Glutamax I (Gibco), N2 and B27 supplements, neurotrophic factor (NF) NGF (R & D Systems), BDNF and GDNF (10 ng / ml each) for neuronal maturation The cell mass was transferred onto an ultra-low attachment dish or multi-wall plate containing. The medium was changed every 3-4 days. 14 days after NF treatment, the cell masses were dissociated by 0.05% trypsin and 10 μg / mL DNaseI followed by gentle pipetting. 10% (volume / volume) FBS (Hyclone), 1 × Glutamax I (Gibco), NF (10 ng / mL each) on a growth factor reduced Matrigel matrix-coated culture plate or glass bottom dish (Matsunami Glass Industry Co., Ltd.) ) And 20 μM Y27632 were added to dissociated cells in DMEM (Nacalai Tesque). Thereafter, the cultures were fed every two days by replacing half of the media with media without Y27632.
Images of cultured cells were acquired using a BZ-X700 fluorescence microscope (Keyence Corporation).

Flow cytometry analysis and FACS
PE-conjugated mouse anti-CD49d antibody (BioLegend), Alexa Fluor488 rat anti-GFP antibody (BioLegend) and Alexa Fluor647 mouse anti-class III beta tubulin (TUBBIII) antibody (BD Biosciences) were used according to the manufacturer's protocol. Flow cytometry was performed using a MACSQuant Analyzer 10 (Miltenyi Biotech). FACS was performed by BD FACSAria II (BD Bioscience). Isotype controls were used as a control population in all experiments.

RNA isolation and RT-PCR
Total RNA extraction from the cells was performed using RNeasy Mini kit (QIAGEN). Total RNA (1 μg) was used for reverse transcription using PrimeScript RT Master Mix (TaKaRa). RT-PCR was performed using Ex Taq Hot Start version (TaKaRa) or PrimeStar GXL DNA polymerase (TaKaRa).

Immunocytochemistry and microscopy Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized in 0.2% Triton X-100 for 10 minutes at room temperature. The cells are then incubated with Block Ace (DS Pharma Biomedical) to prevent any non-specific binding and then incubated with the primary antibody for 12 hours at 4 ° C. or 2 hours at room temperature. I left it alone. Isothermal with a secondary antibody using an appropriate species-specific antiserum bound to either FITC, Alexa647, Cy-3 (Jackson ImmunoResearch; 1/200) or Alexa555 (Invitrogen; 1/1000) The standing was carried out for 1 hour. After staining cell nuclei with DAPI (Sigma Aldrich; 1/1000), cell images were obtained using FV1000 or FV10i confocal microscope (Olympus Corporation). All antibodies were diluted in Block Ace. The following primary antibodies were used at the indicated concentrations: chicken anti-GFP (Abcam; 1/5000), goat anti-PHOX2B (Santa Cruz; 1/200), goat anti-ChAT (Millipore; 1/200) Mouse anti-TUBBIII (Biolegend; 1/1000), rabbit anti-SOX10 (Abcam; 1/200), mouse anti-HOXB7 (R & D Systems; 1/50), rabbit anti-TH (Millipore; 1/1000), Rabbit anti-DBH (Immunostar; 1/400), goat anti-PRPH (Santa Cruz; 1/200), mouse anti-α smooth muscle actin (Abcam; 1: 400).

Quantification of immunocytochemical analysis results Sample images were acquired with the same gain and exposure settings. DAPI ⁺ cells, PHOX2B ⁺ cells, SOX10 ⁺ cells, eGFP ⁺ cells, HOXB7 ⁺ cells or the 9 fields per preparation using ImageJ software program to calculate the average number of TH ⁺ cells measured automatically. Nine visual fields were manually counted per preparation to calculate the average number of ChAT ⁺ cells, PRPH ⁺ cells, or DBH ⁺ cells.

Norepinephrine quantification Norepinephrine concentration in the culture supernatant was measured as reported in Cell Stem Cell 19, 95-106 (2016). The cultured SN was cultured for 15 minutes using HBSS (Gibco). The medium was collected as a control. The cells were cultured for an additional 15 minutes in HBSS containing 50 mM KCl, after which the medium was collected. After media collection, the media was centrifuged at 300 g for 5 minutes to eliminate cells or cell debris. To prevent degradation of norepinephrine, 1 mM EDTA (Gibco) and 4 mM sodium metabisulfite (Nacalai Tesque) were added to the samples and the samples were stored at −80 ° C. until analysis. Total norepinephrine levels in the samples were quantified using an epinephrine / norepinephrine ELISA kit (Abnova) according to the manufacturer's instructions. The norepinephrine release level was determined by subtracting the calculated epinephrine level in the control sample from the epinephrine level in the sample treated with 50 mM KCl.

Statistical analysis The Microsoft Excel 2013 software program was used for statistical analysis. Results are expressed as mean ± standard error (SEM). Statistical significance was determined using Student's t test. “N” represents the number of independent experiments.

<Result>
It was decided to track PHOX2B expression during differentiation using a reporter. PHOX2B is an essential transcription factor for the development of autonomic nervous system neural crest derivatives such as sympathetic ganglia, parasympathetic ganglia and intestinal ganglia in mice. In view of long-lasting PHOX2B expression (from sympathetic NCC to mature neurons), it was hypothesized that tracking PHOX2B expression may be useful in optimizing differentiation protocols. PHOX2B :: eGFP targeting the 3′UTR region of the PHOX2B locus from two types of hPSC clones, namely human embryonic stem cells (hESC, cell line: KhES1) and human induced pluripotent stem cells (hiPSC, cell line: 409B2) A knock-in reporter strain was generated (FIGS. 5a, b).

SN is derived from trunk NCC, and trunk NCC is derived from NMP. NMP is amphoteric for the posterior neural plate and paraxial mesoderm during embryonic body axis development. WNT-mediated retroversion of hPSC is important for NMP induction. The effect of WNT activator CHIR99021 during the first 3 days of agglutination culture was first evaluated. Treatment with 1.5 μM or more of CHIR was effective in increasing the expression of the Hox gene. This indicates that cells under these conditions began to retrograde during 3 days of initial differentiation (FIG. 6a). The NMP markers BRACURY and SOX2 were also expressed under these conditions, confirming that the CHIR-treated aggregate on the third day had the characteristics of NMP. Since expression of the mesoderm-specific transcription factor TBX6 was increased, treatment with a higher dose of CHIR (5 μM) is likely to lead hPSCs to developmental fate to the mesoderm.

In the posterior region of the body, various subtypes of neural progenitor cells (NPCs) and NCC are generated from the NMP-derived neural plate via dorsoventral axis determination. Therefore, we modified the dorsal ventral axis of these day 3 aggregates. Since bone morphogenetic protein (BMP) and sonic hedgehog (SHH) signals are important for dorsalization and abdominalization, respectively, BMP4 and the SHH agonist purmorphamine (Pur) are 1.5 μM. Added to day 3 aggregates treated with 0 μM or 3.0 μM CHIR (FIG. 1 a). In addition, retinoic acid (RA) is effective in inducing NPC via NMP, and neuronal induction of primary neural crest stem cells from extraneural tubes or embryonic autonomic ganglia has been performed in the presence of RA. RA was used in this situation. Under some conditions, PHOX2B :: eGFP ⁺ cells were detected with a purity of over 40% (FIGS. 1b and 6b). Of these, the following ^{(i) CHIR 1.5μM Pur + RA} 1000nM, (ii) CHIR 2.0μM Pur + RA 1000nM, (iii) CHIR 1.5μM BMP + RA 100nM and ^{(iv) CHIR} 2.0μM BMP ⁺ Focused on 4 conditions of RA ^{1000 nM} . The 3.0 μM CHIR treatment detected eGFP ⁺ cells at a relatively low frequency (0-10%) compared to 1.5 μM or 2.0 μM CHIR treatment (data not shown).

PHOX2B is expressed not only in autonomic crest derivatives but also in the central nervous system (CNS) neurons and their neural progenitors in the hindbrain. Since CD49d (integrin α4) is expressed in migratory NCC and their derivatives, CD49d was used to distinguish NCC from other lineages such as NPC in the CNS. More CD49d ⁺ cells were detected under BMP treatment conditions (conditions (iii) and (iv)) than under Pur treatment conditions (conditions (i) and (ii)) (FIG. 1c). Only BMP4-treated cells (conditions (iii) and (iv)) expressed NCC markers SOX10 and FOXD3, whereas Pur-treated cells expressed NPC markers SOX1 and PAX6 more strongly (FIG. 1d). ). Interestingly, in our experiments, RA was important for increasing PHOX2B expression not only under Pur treatment but also under BMP4 treatment (FIGS. 1b, 6b and 7a). Although even without an RA is SOX10 ⁺ cells were detected SOX10 ⁺ and PHOX2B ⁺ double positive cells were found only under conditions comprising RA (Fig. 7b, c). This indicates that BMP4 is involved in NCC induction, and RA modifies the fate of NCC to move toward the autonomous lineage.
It has been described in several previous reports that the level of longitudinal determination is determined by the amount of WNT signal. Consistent with this, in our experiments, the higher the CHIR concentration, the more backward the cells were defined. CHIR treatment at 1.5 μM assigns cells to the hindbrain and cervical spinal cord region (HOXB4 ⁺ HOXB8 ⁺ HOXC9 ⁻ ), while 2.0 μM CHIR treatment causes cells to move from the cervical to thoracic spinal cord region (HOXB4 ⁻ HOXB8 ⁺ HOXC9 ⁺ ) (FIG. 1d).

Under Pur conditions, the majority of eGFP ⁺ cells did not express CD49d (FIGS. 1c and 6c). Since SHH signals ventralize neuroepithelial cells, it was hypothesized that, under conditions (i, ii), eGFP ⁺ cells were progenitors of cranial motor neurons in the ventral posterior brain. In fact, these CD49d ⁻ eGFP ⁺ cells differentiated into neurons expressing choline acetyltransferase (ChAT), a motor neuron marker (FIG. 6d, e).

Thus, 1) aggregates on day 3 treated with CHIR can give rise to both CNS neural progenitor cells and NCC, 2) that BMP treatment and RA treatment are essential for induction of PHOX2B expressing NCC, and 3) It was confirmed that the longitudinal axis at the trunk level was determined for hPSC by the 2.0 μM CHIR treatment. eGFP ⁺ cells actually expressed PHOX2B protein under these conditions, which excluded the possibility of leakage of our reporter system (FIG. 5c).

In order to confirm the induction of sympathetic NCC in our culture, the characteristics of CD49d ⁺ eGFP ⁺ cells under CHIR ^{2.0 μM} BMP ⁺ RA ^{100 nM} conditions were then analyzed (FIG. 2a, b). During differentiation, CD49d ⁺ eGFP ⁺ cells appeared after day 8, and some eGFP ⁺ cells lost CD49d expression after day 10 (FIGS. 8a, b). This suggests that cells at different stages coexist. As expected, among eGFP ⁺ cells, the majority of CD49d ⁺ cells expressed the neuronal marker TUBBIII weakly, while CD49d ⁻ cells expressed TUBBIII strongly (FIG. 8c, d). This indicates that CD49d ⁻ eGFP ⁺ cells are in the late stage of neuronal differentiation lineage determination. The majority of CD49d ⁺ eGFP ⁺ cells were double positive for SOX10 and PHOX2B (FIGS. 2c, d and 8e). This suggests that these cells correspond to double positive progenitor cells of SOX10 and PHOX2B that are localized in mouse embryonic sympathetic ganglia. Since the majority of CD49d ⁺ eGFP ⁺ cells express HOXB7 (FIGS. 2e, f and 8f), these cells were defined on the anterior-posterior axis at the trunk level. In summary, the expression of essential transcription factors in CD49d ⁺ eGFP ⁺ cells under CHIR ^{2.0 μM} BMP ⁺ RA ^{100 nM} conditions is confirmed to be equivalent to the expression of essential transcription factors in mouse sympathetic nervous system NCC in vivo and in vitro. It was done.

Next, CD49d ⁺ eGFP ⁺ cell culture conditions were optimized for SN induction. SN maintained PHOX2B expression throughout differentiation, whereas sympathetic NCC-derived non-neuronal cells lost PHOX2B expression, so eGFP expression was followed again. Because the neurosphere culture method can selectively propagate sympathetic NCC in embryonic mouse sympathetic ganglia in vitro, CD49d ⁺ eGFP ⁺ sympathetic NCC enrichment selected using EGF and FGF2 Cells were cultured in suspension to form aggregates (FIG. 3a). However, about half of those cells lost eGFP expression during the first 7 days of culture after sorting (Figure 3b, c). In animal experiments in vivo and in vitro, BMP4 is required to determine the initial neuronal differentiation lineage of the sympathetic nervous system NCC. Therefore, BMP4 was added and the expression of eGFP was maintained in the majority of cells (> 90%) by the BMP4. Furthermore, when the cells were cultured longer (up to 28 days after sorting), this condition expanded the cell number by more than 10-fold without losing eGFP expression (FIG. 3d).
During the prolonged agglutination culture, the expression of SOX10 declined rapidly, with few cells expressing SOX10 after 14 days (FIGS. 3d and 9b). Considering that maintenance of PHOX2B along with the loss of SOX10 leads to neuronal differentiation lineage determination, neurosphere cultures using BMP4 appear to selectively proliferate sympathetic NCC, Promotes the development of sympathetic nervous system NCC to nerve cells. In fact, the use of BMP4 increased the number of TUBIIIB ⁺ cells during the first 14 days of culture (FIGS. 9c, d). In adhesion culture, most cells lost eGFP expression even in the presence of BMP4, and some cells expressed α-smooth muscle actin, a marker for NC-derived myofibroblasts (FIG. 9e). This shows that suspension culture of aggregates is important for the determination of the neuronal lineage. In summary, we have succeeded in establishing a culture system that proliferates sympathetic lineage cells while maintaining PHOX2B expression.

Next, SN was induced from the neurosphere culture, and the purity of those SNs was evaluated. The seeded BMP4 treated aggregates did not show neuronal morphology (Fig. 9f), probably due to their immaturity. Sympathetic neurons, except for EGF, FGF2 and BMP4, which are all reported to be involved in the determination of the initial neuronal differentiation lineage of SN due to the maturation of neural progenitor cells, but are not sufficient for the induction of mature neurons NGF, BDNF, and GDNF, which are neurotrophic factors (NF) that promote development, were added (FIG. 3e). After 14 days of culture using this modified protocol, the cells exhibited axonal morphology (FIG. 9f). At this point, the majority of those cells (greater than 85%) are tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), both of which are catalyzed enzymes of catecholamine synthesis and thus are markers of noradrenergic neurons Positive (Fig. 3f, g). These cells also expressed peripherin (PRPH), a peripheral nerve cell specific intermediate filament. Therefore, these neurons are peripheral (PRPH ⁺ ) noradrenergic (TH ⁺ DBH ⁺ ) neurons derived from trunk NCC and correspond to SN. This maturation step was applicable to neurosphere cells at various times in culture after sorting (FIGS. 10a-c). Therefore, most PHOX2B expressing neurosphere cells give rise to terminally differentiated neurons when the culture is extended (28 days). Furthermore, neurosphere cells can be cryopreserved by common methods without loss of viability or neuronal differentiation potential (FIGS. 10d-g). Finally, the release of norepinephrine, an SN neurotransmitter, was confirmed as a functional embodiment of these neurons (FIG. 3h). The amount of norepinephrine released from hPSC-derived SN was comparable to that in previous reports.

HPSC was successfully differentiated into SN in stages by improving the culture conditions. However, such differentiation was only possible when using the PHOX2B :: eGFP reporter hPSC line, since a cell sorting step is required for the purification of PHOX2B expressing NCC. Since our ultimate goal is to develop a robust and universal differentiation method that can be applied to various hPSC strains, we next tried to apply our system to hPSC strains without the PHOX2B reporter It was.

For this purpose, the same method was repeated with the exception of cell sorting using the PHOX2B :: eGFP hPSC strain (FIG. 4a). Surprisingly, eGFP ⁺ cells were purified with high frequency (day 17; 75% -85%) after transfer to neurosphere culture, and high frequency (day 31; 75%) after neuronal maturation steps. Time course analysis showed that it was maintained at (˜90%) (FIGS. 4b, c). Immunostaining revealed PHOX2B ⁺ TH ⁺ DBH ⁺ PRPH ⁺ peripheral noradrenergic neurons in 75% -80% of those cells (FIGS. 4d, e). This indicates that the protocol can selectively propagate sympathetic NCC and their derivatives without cell sorting.

For further proof of this principle, the same method was performed using four different hPSC strains (KhES1 and KhES3 strains as hESC strains, 409B2 and 604A1 strains as hiPSC strains) without the PHOX2B :: eGFP reporter. did. In this experimental series, selective growth in neurosphere cultures produced both SOX10 ⁻ PHOX2B ⁺ sympathetic progenitor cells and SN firmly with a purity of 70% to 80% (FIGS. 4f-h). These data demonstrate the certainty of our protocol for the generation of SN from various hPSC strains.

Claims (16)

A method for producing neural crest cells, comprising a step of culturing pluripotent stem cells in a culture solution containing FGF (Fibroblast growth factor) 2, retinoic acid and BMP (Bone morphogenetic protein) 4. The neural crest cell according to claim 1, wherein the concentration of FGF2 is 1 ng / ml to 100 ng / ml, the concentration of retinoic acid is 10 nM to 10 μM, and the concentration of BMP4 is 5 ng / ml to 150 ng / ml. Manufacturing method. Pluripotent stem cells are cultured in a culture solution containing GSK (Glycogen synthase kinase) 3β inhibitor and TGF (Transforming growth factor) β inhibitor, and then cultured in a culture solution containing FGF2, retinoic acid and BMP4. The method for producing a neural crest cell according to claim 1 or 2. The method for producing neural crest cells according to any one of claims 1 to 3, wherein the culture is performed by suspension culture. The method for producing neural crest cells according to any one of claims 1 to 4, comprising a selection step using CD49d. The method for producing neural crest cells according to any one of claims 1 to 5, wherein the pluripotent stem cells are induced pluripotent stem cells. A method for producing sympathetic progenitor cells, comprising culturing neural crest cells in a culture solution containing cytokine and BMP4 to induce sympathetic progenitor cells. The method for producing sympathetic progenitor cells according to claim 7, wherein the cytokines are EGF (Epidermal growth factor) and FGF2. The concentration of BMP4 is 5 ng / ml to 150 ng / ml, the concentration of EGF is 1 ng / ml to 100 ng / ml, and the concentration of FGF2 is 1 ng / ml to 100 ng / ml. A method for producing sympathetic progenitor cells. The method for producing sympathetic progenitor cells according to any one of claims 7 to 9, wherein the culturing is performed in suspension culture. The method for producing sympathetic neural progenitor cells according to any one of claims 7 to 10, wherein the neural crest cells are obtained by the method according to any one of claims 1 to 6. A neural crest cell is produced by the method according to any one of claims 1 to 6, and a sympathetic neural progenitor cell is produced by the method according to any one of claims 7 to 10 using the obtained neural crest cell. For producing sympathetic progenitor cells. A process for producing sympathetic progenitor cells by the method according to any one of claims 7 to 12, and sympathetic progenitor cells obtained by culturing the obtained sympathetic progenitor cells in a culture medium containing neurotrophic factor. A method for producing a sympathetic nerve cell, which comprises a step of maturing a sympathetic nerve cell. The method according to claim 13, wherein the neurotrophic factor is NGF (Nerve growth factor), BDNF (Brain-derived neurotrophic factor) and GDNF (Gliallicell line-derived-neurotrophic factor). A sympathetic nerve cell obtained by the method according to claim 13 or 14. A composition for treating sympathetic neuropathy comprising sympathetic nerve cells obtained by the method according to claim 13 or 14.

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