WO2023238932A1 - 未分化多能性幹細胞の検出方法および検出試薬 - Google Patents

未分化多能性幹細胞の検出方法および検出試薬 Download PDF

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WO2023238932A1
WO2023238932A1 PCT/JP2023/021545 JP2023021545W WO2023238932A1 WO 2023238932 A1 WO2023238932 A1 WO 2023238932A1 JP 2023021545 W JP2023021545 W JP 2023021545W WO 2023238932 A1 WO2023238932 A1 WO 2023238932A1
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cells
pluripotent stem
stem cells
mir302chg
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健二 長船
啓 辻本
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Rege Nephro Co Ltd
Kyoto University NUC
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Rege Nephro Co Ltd
Kyoto University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • renal progenitor cells nephron progenitor cells
  • human pluripotent stem cells such as human iPS cells (iPSCs) and human ES cells
  • iPSCs human iPS cells
  • ES cells iPS cells
  • cell preparations produced from iPS cells have considerable problems with tumorigenicity due to residual iPS cells. Therefore, it is essential to confirm the absence of residual iPS cells and ensure the safety of cell preparations.
  • the cell population is nephron progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteroblasts, interstitial progenitor cells, vascular endothelial cells, chondrocytes, bile duct progenitor cells, and erythropoietin-producing cells induced to differentiate from pluripotent stem cells. , nerve cells, cardiomyocytes, and/or lung cells, or cells obtained at an intermediate stage of differentiation from pluripotent stem cells to any of these cells, the method according to any one of [1] to [11]. .
  • the oligonucleotide primers of (i) are SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: An oligonucleotide primer having one or more combinations of sequences selected from the group of combinations consisting of SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and SEQ ID NO: 32 and SEQ ID NO: 33.
  • an oligonucleotide primer set or the combination of the primer and probe in (ii) above has a sequence that is a combination of SEQ ID NO: 34 and SEQ ID NO: 35; and an oligonucleotide probe comprising the nucleotide sequence set forth in SEQ ID NO: 36.
  • undifferentiated pluripotent stem cells remaining in a cell population containing nephron progenitor cells induced to differentiate from pluripotent stem cells can be detected. This makes it possible to evaluate the safety of regenerative medicine products (for cell therapy, transplant organ reconstruction, etc.) containing nephron progenitor cells derived from human iPS cells. Furthermore, it is expected that residual undifferentiated pluripotent stem cells can be detected in other differentiated cell culture systems other than nephron progenitor cells.
  • TPM values of three splice variants of MIR302CHG, variant ENST00000510655.1, variant ENST00000509938.1, and variant ENST00000505215.1 in human ESCs or human ESC-derived lung progenitor cells or lung organoids.
  • the cell population includes cells that are in the middle of differentiation into nephron progenitor cells (progenitor cells), and nephron progenitor cells obtained by inducing differentiation of pluripotent stem cells or in the process of differentiation from pluripotent stem cells to nephron progenitor cells. Even more preferred is a cell population comprising cells obtained in a step.
  • methods for inducing differentiation of nephron progenitor cells from pluripotent stem cells include, for example, Tsujimoto, H. et al. (2020), Cell Rep. 31, 107476, WO2020/213734, Morizane, R. et al. (2015) Nat. Biotechnol. 33, 1193-1200, Li, Z. et al. (2016) Cell Stem Cell 19, 516-529, 2016 Methods include: As a method for inducing differentiation of pancreatic progenitor cells from pluripotent stem cells, specifically, for example, the method described in Kimura, A. et al., (2020) Cell Chem. Biol. 27, 1561-1572.e7. can be mentioned.
  • vascular endothelial cells and chondrocytes from pluripotent stem cells for example, Tsujimoto H, et al. Cell Rep. 2020 Apr 7;31(1):107476. doi: 10.1016/j
  • Examples include the method described in .celrep.2020.03.040.
  • a method for inducing differentiation of stromal progenitor cells from pluripotent stem cells for example, WO/2023/017848, Takasato M, et al. Nature. 2015;526: 564-568. doi:10.1038/nature15695 Examples include the method described in .
  • measuring the amount of RNA means measuring the amount of RNA, and even if the measured amount is below the detection limit, it is included in “measuring the amount of RNA”. It will be done.
  • the method for measuring the amount of RNA is not particularly limited as long as it is a method that can measure the amount of RNA contained in a cell population, and any known method can be used. Examples include a measurement method using probe hybridization, a measurement method using nucleic acid amplification, a measurement method using mass spectrometry, and a measurement method using sequencing.
  • Measurement methods using nucleic acid amplification include, for example, a method of confirming amplification by quantitative RT-PCR that combines reverse transcription (RT) reaction and PCR, or a method that combines reverse transcription (RT) reaction and 2-step ddPCR.
  • methods include 2-step (RT) ddPCR to confirm amplification, and one-step ddPCR to directly confirm RNA amplification.
  • the method of measuring the amount of RNA of the present invention preferably includes a method of confirming amplification property by quantitative RT-PCR or a method of confirming amplification property by 2-step (RT) ddPCR.
  • RNA is extracted from a cell population
  • DNA is synthesized from the extracted RNA by reverse transcription reaction, and amplification is confirmed by quantitative PCR, or amplification is confirmed by 2-step ddPCR.
  • this is carried out by
  • RNA extracted from a cell population is preferably subjected to a process to remove DNA.
  • the treatment for removing DNA can be appropriately selected from known treatment methods, and includes, for example, a treatment method in which DNA degrading enzyme (DNase) is added.
  • DNase DNA degrading enzyme
  • a method for confirming amplification by quantitative PCR can be appropriately selected from known methods, such as the SYBR (trademark) Green method, the TaqMan (trademark) method, and the like.
  • SYBR trademark
  • TaqMan trademark
  • the above-mentioned process of synthesizing DNA from RNA by reverse transcription reaction and quantitative PCR, which are performed sequentially or simultaneously, is also referred to as quantitative RT-PCR.
  • RNA complementary sequence examples include SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:
  • the method for confirming amplification by 2-step ddPCR is carried out by ddPCR using a combination of oligonucleotide primers and oligonucleotide probes. It is preferable to confirm the amplification of RNA expressed from MIR302CHG by 2-step ddPCR.
  • 2-step ddPCR as an example of an oligonucleotide primer and an oligonucleotide probe for amplifying RNA expressed from MIR302CHG, its DNA complementary sequence, or a part of the RNA complementary sequence, the combination of SEQ ID NO: 34 and SEQ ID NO: 35 is used. and an oligonucleotide probe having the sequence set forth in SEQ ID NO: 36.
  • DNA containing the nucleotide sequence set forth in SEQ ID NO: 36 is used as an oligonucleotide probe, and DNA having a sequence that is a combination of SEQ ID NO: 34 and SEQ ID NO: 35 is used as an oligonucleotide probe. It is preferable to use it as an oligonucleotide primer set.
  • the amount of RNA expressed from MIR302CHG is evaluated by the absolute amount (copy number), and it is determined that undifferentiated pluripotent stem cells exist when a certain amount or more, for example, 1 copy or more or 10 copies or more is detected. good.
  • the amount of RNA expressed from MIR302CHG is normalized by the expression amount of a housekeeping gene (e.g., TATA-box binding protein (TBP) gene), and the value (relative amount) is evaluated. It may be determined that undifferentiated pluripotent stem cells exist when the
  • Undifferentiated pluripotent stem cell-specific antigens include proteins, glycopeptides, glycoproteins, glycolipids, sugar chains, etc. that are specific to undifferentiated pluripotent stem cells.
  • Cell surface antigens are preferred, and specific examples include TRA-1-60.
  • Examples of mRNA switches targeting undifferentiated pluripotent stem cell-specific nucleotides include miR-302a response switch (Fujita Y et al. Science Advance 2022 Jan 7;8(1): eabj1793.) .
  • Examples of conditions for proliferation and adhesion specific to undifferentiated pluripotent stem cells include the Essential-8/LN521 culture amplification method (Tano et al., PLoS One.
  • undifferentiated pluripotent stem cells can be detected in products that use cell populations that can contain undifferentiated pluripotent stem cells, such as regenerative medicine products using human iPS cell-derived nephron progenitor cells. This makes it possible to evaluate safety.
  • the cell population determined to not contain undifferentiated pluripotent stem cells by the method of the present invention may be further evaluated for tumor formation in vivo.
  • the present invention also relates to a reagent kit for detecting undifferentiated pluripotent stem cells, which includes a reagent for measuring the amount of RNA expressed from MIR302CHG.
  • the reagent kit of the present invention includes (i) an oligonucleotide primer for measuring RNA expressed from MIR302CHG, or (ii) an oligonucleotide primer and an oligonucleotide probe for measuring RNA expressed from MIR302CHG. Including combinations.
  • the oligonucleotide primers (i) are, for example, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, An oligo having one or more combinations of sequences selected from the group of combinations consisting of SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and SEQ ID NO: 32 and SEQ ID NO: 33.
  • Human iPS cells (human iPSCs) were used as pluripotent stem cells.
  • Human iPSCs were maintained in feederless culture using Stem Fit AK02N medium (Ajinomoto) or Stem Fit AK03N medium (Ajinomoto) on iMatrix-coated cell culture plates.
  • human iPSCs were passaged every 4 to 5 days using 0.5 mM EDTA/PBS (Thermo Fisher Scientific) and regularly inspected for mycoplasma contamination.
  • Nephron progenitor cells Nephron progenitor cells (NPCs) are described by Tsujimoto, H.; et al. (2020), Cell Rep. It was derived from human iPSCs by the method described in 31, 107476. In addition, cells 4 days after starting differentiation from human iPSCs using the above method were also used for analysis as Day 4 cells (D4C).
  • Renal interstitial progenitor cells Renal interstitial progenitor cells (IPCs) were induced to differentiate from human iPSCs by the method described in WO/2023/017848, specifically by the method described below. Human iPSCs were first enzyme-treated with a 1:1 mixture of TrypLE Select Enzyme (Thermo Fisher Scientific) and 0.5 mM EDTA/PBS for 5 minutes, and washed with PBS(-).
  • basal medium serum-free differentiation medium
  • Basal medium consisting of DMEM/F12 Glutamax (Thermo Fisher Scientific), B27 supplement minus vitamin A (Thermo Fisher Scientific), and 0.5 ⁇ Penicillin/Streptomycin.
  • the cells were cultured for 48 hours using a basal medium containing 5 ⁇ M CHIR99021, 30 ng/mL bFGF, 10 ng/mL activin A, and 30 ⁇ M Y-27632. Then a combination of 1 ⁇ M CHIR99021, 10 ⁇ M SB431542, 100 nM RA, 10 ng/mL IL-1b, 500 nM Smoothened (SMO) agonist (SAG) for 48 hours, then 1 ⁇ M CHIR99021, 10 ⁇ M SB431542, 100 nM The cells were treated with a combination of RA and 500 nM smoothed (SMO) agonist (SAG) for 72 hours and cultured.
  • SMO Smoothened
  • Ureteroblasts Ureteroblasts (ND) were described by Mae, SI, et al. Cell Rep, 32 (2020), 10.1016/j. celrep. It was induced from human iPSCs by the method described in 2020.107963.
  • RNA-Seq data that has already been reported and published in a repository was performed using the following steps.
  • transcripts were quantified using Salmon using Homo sapiens GRCh38 cDNA gene annotation (Ensembl). Output from Salmon was processed using the R/Bioconductor package tximport to obtain gene expression values.
  • DEG analysis was performed using iDEP (version 0.92).
  • iDEP enrichment analysis of the top 1,000 DEGs using the k-means method was performed using the biological process of Gene Ontology (GO).
  • GO Gene Ontology
  • Quantitative RT-PCR was performed using the following procedure. Total RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's recommended protocol. cDNA was synthesized from total RNA by performing a reverse transcription reaction using ReverTra Ace (Toyobo) and oligo dT according to a standard protocol. Quantitative PCR was performed using QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) and TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio). Denaturation was performed at 95°C for 30 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 34 seconds. For data analysis of gene expression levels, quantification was performed using the comparative Ct method, and the values were normalized with TBP, a housekeeping gene. PCR was performed at least three times on each sample. Table 1 shows the primers used for PCR.
  • 2-step ddPCR 2-step ddPCR was performed using the following procedure.
  • Total RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's recommended protocol.
  • cDNA was synthesized from total RNA by performing a reverse transcription reaction using ReverTra Ace (Toyobo) and oligo dT according to a standard protocol.
  • cDNA obtained from total RNA using ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 900 nM forward and reverse primers, and 250 nM probes labeled with FAM or HEX dye and ZEN-Iowa Black fluorescence quencher. was added to perform ddPCR.
  • Cell enrichment using magnetic beads was performed using MACS (Miltenyi Biotec). Human iPSCs and NPCs were dissociated with Accumax (Innovative Cell Technologies, Inc.), and each cell suspension was centrifuged at 200 g for 7 min at 4°C. After centrifugation, the supernatant was discarded and each cell pellet was resuspended in MACS Rinsing solution (Miltenyi Biotec) supplemented with 0.5% BSA (Wako or Miltenyi Biotec) and 10 ⁇ M Y-27632.
  • the positive fraction was flushed out and collected by gently pressing the syringe at a rate of 2-3 drops per second. After centrifugation at 200 g for 5 minutes, cells from the positive and negative fractions were respectively lysed using RLT buffer (QIAGEN) supplemented with ⁇ -mercaptoethanol and stored at -80°C until analysis.
  • RLT buffer QIAGEN
  • RNA-seq analysis including non-coding RNA and found 26 extremely highly expressed genes (log2FC ⁇ - 10 & FDR ⁇ 10 -5 ) (Fig. 1).
  • MIR302CHG which is related to lncRNA, is more effective than LIN28A, which is conventionally used for detecting residual iPS cells, and CUZD1, which is found to be differentially highly expressed in human iPSCs (data omitted) in the analysis of deposited RNA-seq data. It was also found that each transcript (transcripts per million: TPM value) showed a high value for each transcript per million total transcripts (Fig. 2).
  • the TBP-normalized MIR302CHG expression pattern was better at detecting iPSCs than the CUZD1 and POU5F1 expression patterns when the proportion of iPSCs was low.
  • iPSC contamination approximately 10 -3 % (1 cell in 10 5 cells) in NPC and 10 -1 (1 cell in 10 3 cells) in D4C. It was suggested that it could be done.
  • Example 2 Detection of pluripotent stem cells by cell enrichment and quantitative PCR iPSCs representing 10 -4 % of the total number of cells were mixed with the nephron progenitor cell (NPC) cell population to yield 10 -4 % iPSCs.
  • NPC nephron progenitor cell
  • Cell enrichment of 10 -4 % iPSCs using magnetic beads was performed using TRA-1-60 surface antigen as an indicator, and TRA-1-60 positive fraction (PF) and negative fraction (NF) were collected.
  • MIR302CHG For MIR302CHG, CUZD1, and POU5F1, amplification was confirmed by quantitative RT-PCR in each compartment of NPC, iPSC, 10 ⁇ 4 % iPSC, TRA-1-60 PF, and TRA-1-60 NF.
  • MIR302CHG primers having the combination of SEQ ID NOs: 6 and 7 (corresponding to #2 in Table 1 above) were used. The results are shown in FIG.
  • By a combination of enrichment using magnetic beads and quantitative RT-PCR that measures the amount of RNA expressed from MIR302CHG it is possible to detect contamination of 10 -4 % of NPCs or D4C, that is, one or more iPSCs in every 106 . Conceivable.
  • CUZD1 and POU5F1 it was difficult to detect contamination of more than 1 in 10 6 iPSCs in either NPC or D4C, or both, even with a similar method.
  • RNA expressed from MIR302CHG was higher than that of NPCs when 10 -4 % or more of iPSCs were included, and in particular, it was clearly higher when 10 -2 % or more of iPSCs were included. Therefore, by 2-step ddPCR that measures the amount of RNA expressed from MIR302CHG, it is possible to detect contamination of 10 -4 % to 10 -2 % of NPCs, that is, one or more iPSCs in 10 to 104 . Conceivable.
  • Example 4 Expression of MIR302CHG in human iPSC-derived pancreatic progenitor cells and human iPSC-derived hepatocytes
  • Figure 9 is from Kimura et al (2020), Cell Chem. Biol. 27, 1561-1572.
  • Figure 10 shows human iPSCs or human iPSC-derived pancreatic progenitor cells reported in Kotaka, M. et al., 2017, Sci. Rep. 7, 1-13.
  • Example 5 Expression of MIR302CHG in human iPSC-derived neural progenitor cells and neurons was analyzed the expression of MIR302CHG in human iPSC-derived neural progenitor cells and neurons from publicly available RNA-seq datasets.
  • Figure 11 shows the published RNA of human iPSCs or human iPSC-derived neural progenitor cells and neurons reported in Chen et al., 2013, PLoS One. 2013;8: e75682. doi:10.1371/journal.pone.0075682.
  • Example 8 Expression of MIR302CHG in human iPSC, IVF-ESC, or nt-ESC-derived vascular endothelial cells From publicly available RNA-seq datasets, MIR302CHG in human iPSC, IVF-ESC, or nt-ESC and their derived vascular endothelial cells We analyzed the expression of Figure 14 shows human iPSC, IVF-ESC, nt-ESC or human iPSC, IVF-ESC, nt-ESC-derived cells reported in Zhao et. al., Proc Natl Acad Sci U S A. 2017;114: E11111-E11120.
  • TPM values of each transcriptional variant of MIR302CHG analyzed using a publicly available RNA-seq dataset of vascular endothelial cells.
  • High expression of MIR302CHG was observed in iPSCs, but the expression almost disappeared after differentiation. Therefore, even during differentiation into vascular endothelial cells, undifferentiated cells can be detected by MIR302CHG.
  • MIR302CHG by measuring the amount of MIR302CHG, it is possible to detect not only undifferentiated pluripotent stem cells but also cells in the early stages of differentiation such as definitive endoderm and anterior primitive streak, and cells in the foregut, midgut, and hindgut. It was found that it was possible to identify
  • Example 10 Expression of MIR302CHG in Renal Interstitial Progenitor Cells and Ureteroblasts Renal interstitial progenitor cells (IPCs) and ureteroblasts ( ND) were obtained respectively.
  • undifferentiated pluripotent stem cells remaining in a cell population containing nephron progenitor cells induced to differentiate from pluripotent stem cells can be detected. This makes it possible to evaluate the safety of regenerative medicine products (for cell therapy, transplant organ reconstruction, etc.) containing nephron progenitor cells derived from human iPS cells. Furthermore, it is expected that residual undifferentiated pluripotent stem cells can be detected in other differentiated cell culture systems other than nephron progenitor cells.

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