JPWO2020239807A5 - - Google Patents
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- JPWO2020239807A5 JPWO2020239807A5 JP2021570365A JP2021570365A JPWO2020239807A5 JP WO2020239807 A5 JPWO2020239807 A5 JP WO2020239807A5 JP 2021570365 A JP2021570365 A JP 2021570365A JP 2021570365 A JP2021570365 A JP 2021570365A JP WO2020239807 A5 JPWO2020239807 A5 JP WO2020239807A5
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さらに本発明は、脳オルガノイドと共にインビトロ培養するための、本発明によるそのようなミクログリア細胞の使用を対象とする。
[本発明1001]
以下の段階を含む、幹細胞からミクログリアを作製するための方法:
(a) 転写調節因子タンパク質をコードするヌクレオチド配列の、ゲノム上の第1のセーフハーバー部位への標的型挿入;ならびに
(b) 転写因子PU.1のコーディング配列(SEQ ID NO: 1)の、ゲノム上の第2のセーフハーバー部位への標的型挿入であって、該遺伝子が、該転写調節因子タンパク質によって調節される誘導性プロモーターに機能的に連結されている、標的型挿入;PU.1(SEQ ID NO: 2)の発現;ならびに
(c) 増殖因子または小分子の少なくとも1種に曝露しながらの、段階(a)および(b)から得られた幹細胞の培養であって、増殖因子または小分子の該少なくとも1種が、ミクログリアの胚型発達のステージの、または成体型ミクログリアの増殖、分化、もしくは極性化のステージの、少なくとも1つの間のシグナル伝達を再現する、培養。
[本発明1002]
増殖因子または小分子の少なくとも1種が以下からなる群より選択される、本発明1001の方法:アクチビンA(SEQ ID NO: 7)、BMP4(SEQ ID NO: 8)、FGF(SEQ ID NO: 9)、VEGF-A(SEQ ID NO: 10)、LY294002、CHIR99021、SCF(SEQ ID NO: 11)、IL-3(SEQ ID NO: 12)、IL-6(SEQ ID NO: 13)、CSF1(SEQ ID NO: 14)、IL-34(SEQ ID NO: 15)、CSF2(SEQ ID NO: 16)、CD200(SEQ ID NO: 17)、CX3CL1(SEQ ID NO: 18)、TGFβ1(SEQ ID NO: 19)、およびIDE1。
[本発明1003]
増殖因子の少なくとも1種がCSF1(SEQ ID NO: 14)またはIL-34(SEQ ID NO: 15)である、本発明1001または1002の方法。
[本発明1004]
小分子の少なくとも1種がCHIR99021、LY294002、またはIDE1である、前記本発明のいずれかの方法。
[本発明1005]
ゲノム上の第1および第2のセーフハーバー部位が異なる、前記本発明のいずれかの方法。
[本発明1006]
転写因子CEBPBの遺伝子のコーディング配列(SEQ ID NO: 3)の挿入およびその発現をさらに含む、前記本発明のいずれかの方法。
[本発明1007]
転写因子RUNX1の遺伝子のコーディング配列(SEQ ID NO: 4)の挿入およびその発現をさらに含む、前記本発明のいずれかの方法。
[本発明1008]
転写因子IRF8の遺伝子のコーディング配列(SEQ ID NO: 5)の挿入およびその発現をさらに含む、前記本発明のいずれかの方法。
[本発明1009]
転写因子SALL1の遺伝子のコーディング配列(SEQ ID NO: 6)の挿入およびその発現をさらに含む、前記本発明のいずれかの方法。
[本発明1010]
転写調節因子タンパク質がリバーステトラサイクリントランス活性化因子(rtTA)(SEQ ID NO: 20)であり、かつその活性がドキシサイクリンまたはテトラサイクリンによって制御される、前記本発明のいずれかの方法。
[本発明1011]
誘導性プロモーターがTet応答性エレメント(TRE)(SEQ ID NO: 21)を含む、前記本発明のいずれかの方法。
[本発明1012]
ゲノム上の第1および第2のセーフハーバー部位が以下からなる群より選択される、前記本発明のいずれかの方法:hROSA26座位(SEQ ID NO: 22)、AAVS1座位(SEQ ID NO: 23)、CLYBL遺伝子(SEQ ID NO: 24)、CCR5遺伝子(SEQ ID NO. 25)、HPRT遺伝子(SEQ ID NO. 26)、または第8染色体上の部位ID 325(SEQ ID NO: 27)を有する遺伝子、第1染色体上の部位ID 227(SEQ ID NO: 28)を有する遺伝子、第2染色体上の部位ID 229(SEQ ID NO: 29)を有する遺伝子、第5染色体上の部位ID 255(SEQ ID NO: 30)を有する遺伝子、第14染色体上の部位ID 259(SEQ ID NO: 31)を有する遺伝子、X染色体上の部位ID 263(SEQ ID NO: 32)を有する遺伝子、第2染色体上の部位ID 303(SEQ ID NO: 33)を有する遺伝子、第4染色体上の部位ID 231(SEQ ID NO: 34)を有する遺伝子、第5染色体上の部位ID 315(SEQ ID NO: 35)を有する遺伝子、第16染色体上の部位ID 307(SEQ ID NO: 36)を有する遺伝子、第6染色体上の部位ID 285(SEQ ID NO: 37)を有する遺伝子、第6染色体上の部位ID 233(SEQ ID NO: 38)を有する遺伝子、第134染色体上の部位ID 311(SEQ ID NO: 39)を有する遺伝子、第7染色体上の部位ID 301(SEQ ID NO: 40)を有する遺伝子、第8染色体上の部位ID 293(SEQ ID NO: 41)を有する遺伝子、第11染色体上の部位ID 319(SEQ ID NO: 42)を有する遺伝子、第12染色体上の部位ID 329(SEQ ID NO: 43)を有する遺伝子、およびX染色体上の部位ID 313(SEQ ID NO: 44)を有する遺伝子。
[本発明1013]
幹細胞が、多能性幹細胞、人工多能性幹細胞(iPSC)、神経前駆細胞、造血幹細胞、または胚性幹細胞(ESC)である、前記本発明のいずれかの方法。
[本発明1014]
幹細胞がヒトまたはマウスの幹細胞である、前記本発明のいずれかの方法。
[本発明1015]
本発明1001~1014のいずれかの方法によって得られるミクログリアであって、好ましくは、以下からなる群より選択されるミクログリア表面タンパク質のうち少なくとも1種を発現する、ミクログリア:ITGAM (CD11B)(SEQ ID NO: 45)、ITGAX(CD11C)(SEQ ID NO: 46)、CD14(SEQ ID NO: 47)、CD16(SEQ ID NO: 48)、ENTPD1(CD39)(SEQ ID NO: 49)、PTPRC(CD45)(SEQ ID NO: 50)、CD68(SEQ ID NO: 51)、CSF1R(CD115)(SEQ ID NO: 52)、CD163(SEQ ID NO: 53)、CX3CR1(SEQ ID NO: 54)、TREM2(SEQ ID NO: 55)、P2RY12(SEQ ID NO: 56)、TMEM119(SEQ ID NO: 57)、およびHLA-DR(SEQ ID NO: 58)。
[本発明1016]
治療に使用するための、本発明1015のミクログリア。
[本発明1017]
疾患のインビトロ診断のための、本発明1015または1016のミクログリアの使用。
[本発明1018]
疾患が以下からなる群より選択される、本発明1017のミクログリアの使用:中枢神経系の疾患、好ましくは神経変性疾患;より好ましくはアルツハイマー病、パーキンソン病、前頭側頭型認知症、もしくは筋萎縮性側索硬化症;神経炎症性疾患または自己免疫疾患、好ましくは多発性硬化症、自己抗体介在性脳炎、もしくは感染性疾患、神経血管疾患;好ましくは脳卒中、血管炎;外傷性脳損傷、およびがん。
[本発明1019]
脳オルガノイドと共にインビトロ培養するための、本発明1015または1016のミクログリアの使用。
Furthermore, the present invention is directed to the use of such microglial cells according to the invention for in vitro culture with brain organoids.
[Invention 1001]
A method for generating microglia from stem cells comprising the steps of:
(a) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein into a first safe harbor site on the genome; and
(b) targeted insertion of the transcription factor PU.1 coding sequence (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and
(c) culturing the stem cells obtained from steps (a) and (b) while being exposed to at least one growth factor or small molecule, wherein said at least one growth factor or small molecule is microglia; A culture that recapitulates signaling during at least one of the stages of embryonic development of adult microglia, or the stages of proliferation, differentiation, or polarization of adult microglia.
[Invention 1002]
The method of the invention 1001, wherein at least one of the growth factors or small molecules is selected from the group consisting of: activin A (SEQ ID NO: 7), BMP4 (SEQ ID NO: 8), FGF (SEQ ID NO: 9), VEGF-A (SEQ ID NO: 10), LY294002, CHIR99021, SCF (SEQ ID NO: 11), IL-3 (SEQ ID NO: 12), IL-6 (SEQ ID NO: 13), CSF1 (SEQ ID NO: 14), IL-34 (SEQ ID NO: 15), CSF2 (SEQ ID NO: 16), CD200 (SEQ ID NO: 17), CX3CL1 (SEQ ID NO: 18), TGFβ1 (SEQ ID NO: 18) NO: 19), and IDE1.
[Invention 1003]
The method of invention 1001 or 1002, wherein at least one of the growth factors is CSF1 (SEQ ID NO: 14) or IL-34 (SEQ ID NO: 15).
[Invention 1004]
The method of any preceding invention, wherein at least one of the small molecules is CHIR99021, LY294002, or IDE1.
[Invention 1005]
The method of any of the preceding inventions, wherein the first and second safe harbor sites on the genome are different.
[Invention 1006]
The method of any of the preceding claims, further comprising inserting the coding sequence (SEQ ID NO: 3) of the gene for the transcription factor CEBPB and expressing it.
[Invention 1007]
The method of any of the preceding claims, further comprising inserting the coding sequence (SEQ ID NO: 4) of the gene for the transcription factor RUNX1 and expressing it.
[Invention 1008]
The method of any of the preceding claims, further comprising the insertion of the coding sequence (SEQ ID NO: 5) of the gene for the transcription factor IRF8 and expression thereof.
[Invention 1009]
The method of any of the preceding claims, further comprising the insertion of the coding sequence (SEQ ID NO: 6) of the gene for the transcription factor SALL1 and expression thereof.
[Invention 1010]
The method of any of the preceding claims, wherein the transcriptional regulator protein is reverse tetracycline transactivator (rtTA) (SEQ ID NO: 20) and whose activity is regulated by doxycycline or tetracycline.
[Invention 1011]
Any of the preceding methods of the invention, wherein the inducible promoter comprises a Tet responsive element (TRE) (SEQ ID NO: 21).
[Invention 1012]
The method of any of the above inventions, wherein the first and second genomic safe harbor sites are selected from the group consisting of: hROSA26 locus (SEQ ID NO: 22), AAVS1 locus (SEQ ID NO: 23). , the CLYBL gene (SEQ ID NO: 24), the CCR5 gene (SEQ ID NO. 25), the HPRT gene (SEQ ID NO. 26), or the gene with site ID 325 on chromosome 8 (SEQ ID NO: 27) , the gene with site ID 227 (SEQ ID NO: 28) on chromosome 1, the gene with site ID 229 (SEQ ID NO: 29) on chromosome 2, the gene with site ID 255 on chromosome 5 (SEQ ID NO: 29) NO: 30), gene with site ID 259 (SEQ ID NO: 31) on chromosome 14, gene with site ID 263 (SEQ ID NO: 32) on chromosome X, on chromosome 2 Gene with site ID 303 (SEQ ID NO: 33), gene with site ID 231 (SEQ ID NO: 34) on chromosome 4, site ID 315 (SEQ ID NO: 35) on chromosome 5 Gene, gene with site ID 307 (SEQ ID NO: 36) on chromosome 16, gene with site ID 285 (SEQ ID NO: 37) on chromosome 6, site ID 233 on chromosome 6 (SEQ ID NO: 37) ID NO: 38), gene with site ID 311 (SEQ ID NO: 39) on chromosome 134, gene with site ID 301 (SEQ ID NO: 40) on chromosome 7, chromosome 8 Gene with site ID 293 (SEQ ID NO: 41) on chromosome 11, gene with site ID 319 (SEQ ID NO: 42) on chromosome 12, site ID 329 (SEQ ID NO: 43) on chromosome 12 and the gene with site ID 313 (SEQ ID NO: 44) on the X chromosome.
[Invention 1013]
The method of any of the preceding inventions, wherein the stem cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), neural progenitor cells, hematopoietic stem cells, or embryonic stem cells (ESCs).
[Invention 1014]
The method of any of the preceding inventions, wherein the stem cells are human or mouse stem cells.
[Invention 1015]
Microglia obtained by the method of any of the inventions 1001-1014, preferably expressing at least one microglial surface protein selected from the group consisting of: ITGAM (CD11B) (SEQ ID NO: 45), ITGAX (CD11C) (SEQ ID NO: 46), CD14 (SEQ ID NO: 47), CD16 (SEQ ID NO: 48), ENTPD1 (CD39) (SEQ ID NO: 49), PTPRC (CD45 ) (SEQ ID NO: 50), CD68 (SEQ ID NO: 51), CSF1R (CD115) (SEQ ID NO: 52), CD163 (SEQ ID NO: 53), CX3CR1 (SEQ ID NO: 54), TREM2 ( SEQ ID NO: 55), P2RY12 (SEQ ID NO: 56), TMEM119 (SEQ ID NO: 57), and HLA-DR (SEQ ID NO: 58).
[Invention 1016]
Microglia of the invention 1015 for use in therapy.
[Invention 1017]
Use of microglia of the invention 1015 or 1016 for in vitro diagnosis of disease.
[Invention 1018]
Use of microglia according to the invention 1017, wherein the disease is selected from the group consisting of: diseases of the central nervous system, preferably neurodegenerative diseases; more preferably Alzheimer's disease, Parkinson's disease, frontotemporal dementia, or muscle atrophy neuroinflammatory or autoimmune diseases, preferably multiple sclerosis, autoantibody-mediated encephalitis, or infectious diseases, neurovascular diseases; preferably stroke, vasculitis; traumatic brain injury, and cancer.
[Invention 1019]
Use of microglia of the invention 1015 or 1016 for in vitro culture with brain organoids.
Claims (20)
(a) 転写調節因子タンパク質をコードするヌクレオチド配列の、ゲノム上の第1のセーフハーバー部位への標的型挿入;ならびに
(b) 転写因子PU.1のコーディング配列(SEQ ID NO: 1)の、ゲノム上の第2のセーフハーバー部位への標的型挿入であって、遺伝子が、該転写調節因子タンパク質によって調節される誘導性プロモーターに機能的に連結されている、標的型挿入;PU.1(SEQ ID NO: 2)の発現;ならびに
(c) 増殖因子または小分子の少なくとも1種に曝露しながらの、段階(a)および(b)から得られた幹細胞の培養であって、増殖因子または小分子の該少なくとも1種が、ミクログリアの胚型発達のステージの、または成体型ミクログリアの増殖、分化、もしくは極性化のステージの、少なくとも1つの間のシグナル伝達を再現する、培養。 A method for generating microglia from stem cells comprising the steps of:
(a) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein into a first safe harbor site on the genome; and
(b) targeted insertion of the transcription factor PU.1 coding sequence (SEQ ID NO: 1) into a second safe harbor site on the genome , the gene being regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and
(c) culturing the stem cells obtained from steps (a) and (b) while being exposed to at least one growth factor or small molecule, wherein said at least one growth factor or small molecule is microglia; A culture that recapitulates signaling during at least one of the stages of embryonic development of adult microglia, or the stages of proliferation, differentiation, or polarization of adult microglia.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP19176722 | 2019-05-27 | ||
EP19176722.7 | 2019-05-27 | ||
PCT/EP2020/064649 WO2020239807A1 (en) | 2019-05-27 | 2020-05-27 | Rapid and deterministic generation of microglia from human pluripotent stem cells |
Publications (2)
Publication Number | Publication Date |
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JP2022534260A JP2022534260A (en) | 2022-07-28 |
JPWO2020239807A5 true JPWO2020239807A5 (en) | 2023-06-02 |
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JP2021570365A Pending JP2022534260A (en) | 2019-05-27 | 2020-05-27 | Rapid and definitive generation of microglia from human pluripotent stem cells |
Country Status (11)
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US (1) | US20220220441A1 (en) |
EP (1) | EP3976766A1 (en) |
JP (1) | JP2022534260A (en) |
KR (1) | KR20220013556A (en) |
CN (1) | CN114174523A (en) |
AU (1) | AU2020281762A1 (en) |
BR (1) | BR112021023618A2 (en) |
CA (1) | CA3139235A1 (en) |
MX (1) | MX2021014456A (en) |
SG (1) | SG11202112475QA (en) |
WO (1) | WO2020239807A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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EP4368718A1 (en) * | 2021-07-06 | 2024-05-15 | Keio University | Microglial progenitor cells, method for manufacturing microglia, and manufactured microglial progenitor cells and microglia |
WO2023102471A1 (en) * | 2021-12-01 | 2023-06-08 | Yale University | Human cortical organoids with engineered microglia-like cells |
WO2023135318A1 (en) * | 2022-01-17 | 2023-07-20 | Westfälische Wilhelms-Universität Münster | Method for expression of a transgene of interest from neural precursor cells |
JP2023140135A (en) * | 2022-03-22 | 2023-10-04 | 株式会社リコー | Microglia production method |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2016210313A1 (en) * | 2015-06-24 | 2016-12-29 | Whitehead Institute For Biomedical Research | Culture medium for generating microglia from pluripotent stem cells and related methods |
JP7235507B2 (en) * | 2016-03-03 | 2023-03-08 | ニューヨーク ステム セル ファウンデーション インコーポレイテッド | Pluripotent stem cell-derived microglia and methods of making and using the same |
US11345891B2 (en) * | 2017-04-05 | 2022-05-31 | Asgard Therapeutics Ab | Compositions for reprogramming cells into dendritic cells or antigen presenting cells, methods and uses thereof |
WO2018204262A1 (en) * | 2017-05-01 | 2018-11-08 | President And Fellows Of Harvard College | Transcription factors controlling differentiation of stem cells |
CN110205295B (en) * | 2019-06-25 | 2021-10-29 | 中国科学院动物研究所 | Method and kit for inducing pluripotent stem cells to generate microglia |
EP4368718A1 (en) * | 2021-07-06 | 2024-05-15 | Keio University | Microglial progenitor cells, method for manufacturing microglia, and manufactured microglial progenitor cells and microglia |
-
2020
- 2020-05-27 US US17/613,927 patent/US20220220441A1/en active Pending
- 2020-05-27 KR KR1020217040483A patent/KR20220013556A/en unknown
- 2020-05-27 BR BR112021023618A patent/BR112021023618A2/en unknown
- 2020-05-27 CA CA3139235A patent/CA3139235A1/en active Pending
- 2020-05-27 SG SG11202112475QA patent/SG11202112475QA/en unknown
- 2020-05-27 WO PCT/EP2020/064649 patent/WO2020239807A1/en unknown
- 2020-05-27 CN CN202080054019.8A patent/CN114174523A/en active Pending
- 2020-05-27 EP EP20733894.8A patent/EP3976766A1/en active Pending
- 2020-05-27 JP JP2021570365A patent/JP2022534260A/en active Pending
- 2020-05-27 MX MX2021014456A patent/MX2021014456A/en unknown
- 2020-05-27 AU AU2020281762A patent/AU2020281762A1/en active Pending
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