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WO2025061781A1 - Method for the determination of pluripotency of stem cells - Google Patents

Method for the determination of pluripotency of stem cells Download PDF

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Publication number
WO2025061781A1
WO2025061781A1 PCT/EP2024/076141 EP2024076141W WO2025061781A1 WO 2025061781 A1 WO2025061781 A1 WO 2025061781A1 EP 2024076141 W EP2024076141 W EP 2024076141W WO 2025061781 A1 WO2025061781 A1 WO 2025061781A1
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Prior art keywords
cells
ipscs
amplification
gene specific
amplifying
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French (fr)
Inventor
Jochen DOBNER
Jean Krutmann
Andrea Rossi
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Iuf Leibniz Institut Fuer Umweltmedizinische Forschung GmbH
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Iuf Leibniz Institut Fuer Umweltmedizinische Forschung GmbH
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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

Definitions

  • the present invention relates to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs).
  • the present invention further relates to a kit for determining the integrity of iPSCs, to an in vitro method for determining the status of differentiated iPSCs, to an in vitro method for determining the status of iPSCs, especially the differentiation status of iPSCs, and to a computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample.
  • iPSC induced pluripotent stem cell
  • iPSCs Induced pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • iPSCs Human induced pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • stem cell-like features including capability to differentiate into cells of each of the three primary germ layers, endoderm, mesoderm, and ectoderm
  • iPSCs Human iPSCs can be genetically modified, e.g., by CRISPR/Cas, which further increases their use range application and research spectrum (Hockemeyer, D.; Jaenisch, R. Induced Pluripotent Stem Cells Meet Genome Editing. Cell Stem Cell 2016, 18, 573-586).
  • iPSC lines in use need to be quality-checked to ensure their integrity.
  • quality control includes assessment of genome (both nuclear and mitochondrial) integrity, as well as assessment of pluripotency status and differentiation capability (Rossi, A.; Lickfett, S.; Martins, S.; Prigione, A. A Call for Consensus Guidelines on Monitoring the Integrity of Nuclear and Mitochondrial Genomes in Human Pluripotent Stem Cells.
  • RT-qPCR Quantitative real time PCR
  • RT-qPCR Quantitative real time PCR
  • Scorecard approach which was developed to enable detection of germ layer-specific marker gene expressions of EBs (Tsankov, A.M.; Akopian, V.; Pop, R.; Chetty, S.; Gifford, C.A.; Daheron, L.; Tsankova, N.M.; Meissner, A.
  • a QPCR ScoreCard Quantifies the Differentiation Potential of Human Pluripotent Stem Cells.
  • Sekine, K. et al. Robust detection of undifferentiated iPSC among differentiated cells.
  • Scientific Reports 2020, 10, 10293 disclosing that marker genes, which are expressed specifically and highly in undifferentiated iPSC, were selected from single cell RNA sequence data to perform detections of residual undifferentiated cells in differentiated cell products.
  • NGS next generation sequencing
  • S0X2 which is still used as a pluripotency marker but also highly expressed in ectoderm differentiation
  • the object of the present invention is therefore to provide an in vitro method for determining the integrity of iPSCs, a kit for determining the integrity of iPSCs, in vitro methods for determining the status of iPSCs or differentiated iPSCs, and a computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample which are able to overcome at least some of the disadvantages of the prior art.
  • iPSC induced pluripotent stem cell
  • iPSCs induced pluripotent stem cells
  • the method of the present invention allows the determination of the integrity of iPSCs in a convenient, fast, easy-to-use, easy- to-access, and easily quantifiable way, enables unequivocal characterisation of analysed samples, and provides means to screen many samples simultaneously.
  • the method for determining the integrity of iPSCs of the present invention is an in vitro method.
  • the integrity of the iPSCs determined in the method of the invention may be the pluripotency status of the iPSCs and/or the differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm.
  • the pluripotency status indicates whether the iPSCs are pluripotent, i.e., whether they theoretically have the potential to differentiate into any of the three primary germ layers: endoderm (progenitor of gut, lungs, yolk sac), ectoderm (progenitor of nervous, sensory, epidermis), and mesoderm (progenitor of muscle, skeleton, blood vascular, urogenital, dermis).
  • the differentiation capacity indicates whether the iPSCs actually differentiate into the three primary germ layers endoderm, ectoderm, and mesoderm.
  • step a) of the method for determining the integrity of iPSCs some of the iPSCs, i.e., pluripotent stem cells generated directly from a somatic cell, which may also be iPSCs manipulated by genome editing, to be evaluated are differentiated into endodermal, ectodermal, and mesodermal cells.
  • the method of the present invention may comprise the following step a): differentiating the iPSCs into endodermal, ectodermal, and mesodermal cells. Methods for differentiating samples of iPSCs into endodermal, ectodermal, and mesodermal cells are known to the person skilled in the art.
  • a first aliquot of each of the samples prepared in step b) is mixed with a first set of amplification primers capable of amplifying a gene specific for the respective cell type, or a part of said gene specific for the respective cell type.
  • samples from four different cell types plural cells/ iPSCs, endodermal cells, ectodermal cells, mesodermal cells
  • the method of the invention requires at least four mixing steps, which are designated as steps cla), c2a), c3a), and c4a).
  • a first aliquot of the iPSCs-derived sample (i.e., the pluripotent cells-derived sample) is mixed with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part of a gene specific for pluripotent cells, thereby forming an amplification mixture cla).
  • a first aliquot of the endodermal cells-derived sample is mixed with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part of a gene specific for endodermal cells, thereby forming an amplification mixture.
  • a gene specific for pluripotent cells is a gene that is only expressed at high levels in pluripotent cells, but not at high levels in endodermal, ectodermal, and mesodermal cells. Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in pluripotent cells, but not at high levels in endodermal, ectodermal, and mesodermal cells, respectively. Therefore, such a gene may be used to unequivocally determine the differentiation state "pluripotent" (by analysing levels of mRNA molecules).
  • a part of the gene specific for pluripotent cells may, e.g., be a fragment or segment of the gene specific for pluripotent cells.
  • amplification primers capable of amplifying such part of a gene specific for pluripotent cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for pluripotent cells, e.g., by amplifying cDNA.
  • a gene specific for endodermal cells is a gene that is only expressed at high levels in endodermal cells, but not at high levels in pluripotent, ectodermal, and mesodermal cells.
  • Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in endodermal cells, but not at high levels in pluripotent, ectodermal, and mesodermal cells, respectively. Therefore, such gene may be used to unequivocally determine the differentiation state "endodermal” (by analysing levels of mRNA molecules).
  • a part of the gene specific for endodermal cells may, e.g., be a fragment or segment of the gene transcript specific for endodermal cells.
  • amplification primers capable of amplifying such part of a gene specific for endodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for endodermal cells, e.g., by amplifying cDNA.
  • a part of the gene specific for ectodermal cells may, e.g., be a fragment or segment of the gene specific for ectodermal cells. It may be advantageous to use amplification primers capable of amplifying such part of a gene specific for ectodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for ectodermal cells, e.g., by amplifying cDNA.
  • amplification primers capable of amplifying such part of a gene specific for mesodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for mesodermal cells, e.g., by amplifying cDNA.
  • step c2a) is performed before step cla
  • step c3a) is performed before step cla
  • all steps cla), c2a), c3a), and c4a) are performed at the same time.
  • the only requirement is that the steps cla), c2a), c3a), and c4a) are performed after step b) and before step d) of the method of the invention.
  • step d) of the method for determining the integrity of iPSCs the amplification mixtures generated in steps cla), c2a), c3a), and c4a) are processed further.
  • the at least four amplification mixtures obtained in the method of the invention are subjected to amplification reactions and the resulting amplification products are quantified.
  • the amount of the amplification products may be normalised relative to the amount of at least one reference gene, i.e., a gene that is expressed in the target cells (iPSCs, endodermal, ectodermal, and mesodermal cells) at a similar level.
  • Suitable reference genes such as GAPDH, ACTB, PPIA, etc., are known to the person skilled in the art.
  • step e) of the method for determining the integrity of iPSCs according to the present invention the results of the quantification of the amplification products are analysed, e.g., by using a software implemented on a computer, thereby obtaining a score that allows to assess the integrity of the iPSCs.
  • This final score indicating the integrity of the iPSCs is also called "hiPSCore" in the following.
  • At least one gene specific for iPSCs or part thereof, at least one gene specific for endodermal cells or part thereof, at least one gene specific for ectodermal cells or part thereof, and at least one gene specific for mesodermal cells or part thereof is amplified, quantified, and analysed.
  • the quality of the analysis result obtained in step e) of the method of the invention i.e., the quality of the score obtained in step e) of the method of the invention, may be improved by amplifying, quantifying, and analysing more than one gene specific for iPSCs, endodermal cells, ectodermal cells, or mesodermal cells, respectively.
  • the method therefore additionally comprises at least one of the following steps: clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2b) mixing a second aliquot of the endodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3b) mixing a second aliquot of the ectodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4b) mixing a second aliquot of the mesodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent
  • the method of the invention may comprise one of the additional steps clb), c2b), c3b), and c4b), two of the additional steps clb), c2b), c3b), and c4b), three of the additional steps clb), c2b), c3b), and c4b), or all of the four of the additional steps clb), c2b), c3b), and c4b).
  • step clb) is performed after step cla) or before step cla). It is also possible that step clb) is performed after step c4a), and so on. It is also possible that all steps cla), c2a), c3a), c4a), clb), c2b), c3b), and c4b) are performed at the same time.
  • the method therefore additionally comprises at least one of the following steps: clc) mixing a third aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2c) mixing a third aliquot of the endodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3c) mixing a third aliquot of the ectodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4c) mixing a third aliquot of the mesodermal cells-derived sample with a third set of amplification primers capable of amplifying
  • the method of the invention may comprise one of the additional steps clc), c2c), c3c), and c4c), two of the additional steps clc), c2c), c3c), and c4c), three of the additional steps clc), c2c), c3c), and c4c), or all of the four of the additional steps clc), c2c), c3c), and c4c).
  • step clc is performed after step cla), before step cla), after step clb), or before step clb). It is also possible that step clc) is performed after step c4a) or after step c4b), and so on. It is also possible that all steps cla), c2a), c3a), c4a), clb), c2b), c3b), c4b), clc), c2c), c3c), and c4c) are performed at the same time.
  • the present invention refers to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs) comprising the following steps: a) differentiating samples of the iPSCs into endodermal, ectodermal, and mesodermal cells; b) preparing samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells; cla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2a) mixing a first aliquot of the iPSCs into end
  • iPSCs induced pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • the gene specific for mesodermal cells may be at least one selected from the group consisting of HAND1, H0XB7, APLNR, LAPTM5, TNFRSF1B, GJA4, TIE1, ADGRL4, S1PR1, TBX15, PRRX1, ID3, EFNA1, GJ AS, FCN3, PLA2G2A, SHE, GBP2, GBP4, SAM Dll, DYNLT5, ADAMIS, GSTM3, CSRP1, TALI, DDR2, CYP1B1, EPAS1, CRIM1, CHMP3, HNMT, CNRIP1, GYPC, CALCRL, NAB1, COL3A1, DPP4, CYRIA, H0XD13, H0XD11, HOXDIO, H0XD9, COL6A3, CTDSP1, RASGRP3, FRMD4B, ARHGAP31, TM4SF1, TNFSF10, SST, PTH1R, WNT5A, PLSCR5, SEMA3G
  • pluripotent cells i.e., genes listed above under item (i)
  • one gene specific for endodermal cells i.e., genes listed above under item (ii)
  • one gene specific for ectodermal cells i.e., genes listed above under item (iii)
  • mesodermal cells endodermal cells i.e., genes listed above under item (iv)
  • two or more genes for pluripotent cells are amplified, or that three or more genes for pluripotent cells are amplified, respectively.
  • one or two genes specific for pluripotent cells are amplified.
  • genes for endodermal cells ectodermal cells, and mesodermal cells.
  • genes for endodermal cells ectodermal cells, and mesodermal cells.
  • one, two, three or more genes specific for the respective cell type can be amplified.
  • one or two genes are amplified for each of the cell types.
  • the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1.
  • the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1.
  • the gene specific for mesodermal cells is selected from of HAND1, H0XB7, and/or APLNR.
  • the gene specific for pluripotent cells is selected from CNMD, NANOG, and/or SPP1, and/or the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1, and/or the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1, and/or the gene specific for mesodermal cells is selected from of HAND1, H0XB7, and/or APLNR.
  • the gene specific for pluripotent cells is selected from CNMD, NANOG, and/or SPP1, and the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1, and the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1, and the gene specific for mesodermal cells is selected from HANDl, H0XB7, and/or APLNR. It was shown that this combination of genes allows a determination of pluripotency and differentiation capacity of stem cells with high accuracy and high specificity.
  • the in vitro method of the present invention comprises amplification of genes or gene transcripts, respectively, specific for pluripotent cells, amplification of genes or gene transcripts, respectively, specific for endodermal cells, amplification of genes or gene transcripts, respectively, specific for ectodermal cells, and amplification of genes or gene transcripts, respectively, specific for mesodermal cells, or amplification of parts of the aforementioned genes or gene transcripts, respectively.
  • Preferred primers are selected from:
  • the first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 7 and 8, the pair SEQ ID NO: 9 and 10, and the pair SEQ ID NO: 11 and 12; and/or (Hi) the first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 13 and 14, the pair SEQ ID NO: 15 and 16, and the pair SEQ ID NO: 17 and 18; and/or
  • the first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 19 and 20, the pair SEQ ID NO: 21 and 22, and the pair SEQ ID NO: 23 and 24.
  • the preferred amplification primers according to SEQ ID NO: 1 and 2 enable for amplification of CNMD.
  • the preferred amplification primers according to SEQ ID NO: 3 and 4 enable for amplification of NANOG.
  • the preferred amplification primers according to SEQ ID NO: 5 and 6 enable for amplification of SPP1.
  • the preferred amplification primers according to SEQ ID NO: 7 and 8 enable for amplification of GATA6.
  • the preferred amplification primers according to SEQ ID NO: 9 and 10 enable for amplification of EOMES.
  • the preferred amplification primers according to SEQ ID NO: 11 and 12 enable for amplification of CER1.
  • the preferred amplification primers according to SEQ ID NO: 13 and 14 enable for amplification of PAX6.
  • the preferred amplification primers according to SEQ ID NO: 15 and 16 enable for amplification of HES5.
  • the preferred amplification primers according to SEQ ID NO: 17 and 18 enable for amplification of PAMR1.
  • the preferred amplification primers according to SEQ ID NO: 19 and 20 enable for amplification of HANDl.
  • the preferred amplification primers according to SEQ ID NO: 21 and 22 enable for amplification of H0XB7.
  • the preferred amplification primers according to SEQ ID NO: 23 and 24 enable for amplification of APLNR.
  • the differentiation of iPSCs in step a) is effected via directed differentiation. Methods of performing directed differentiation are known to the person skilled in the art.
  • the samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells of step b) may be total RIMA isolated from the respective cells or molecules derived from total RNA isolated from the respective cells (e.g., cDNA).
  • the samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells of step b) are preferably cDNA samples, preferably obtained by reverse transcription reaction from RNA isolated from the respective cells. Methods of isolating RNA from eukaryotic cells as well as methods of performing reverse transcription reactions of RNA are known to the person skilled in the art.
  • amplification reactions performed in step d) of the method according to the present invention can be performed by any method known by the skilled artisan. They are preferably PCR methods such as quantitative polymerase chain reactions (qPCR) or digital polymerase chain reactions (dPCR).
  • qPCR quantitative polymerase chain reactions
  • dPCR digital polymerase chain reactions
  • step dPCR is based on conventional end-point PCR coupled with fluorescence intensity measurement after the reaction is completed.
  • the results obtained from step d) are further analysed in step e) of the method according to the present invention.
  • the analysis allows to obtain a score, which ranges from 0 to 4. A score of 0 would indicate that the iPSCs have a bad integrity, e.g., are already differentiated and/or display impaired differentiation capacity.
  • kits for determining the integrity of iPSCs comprising a) a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof; b) a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof; c) a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof; d) a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof.
  • the amplification primers are preferably those described above for each of the different cell types.
  • the object of the present application is further solved by an in vitro method for determining the status of differentiated iPSCs comprising the following steps: a) providing a differentiated iPSCs-derived sample; bl) mixing a first aliquot of the differentiated iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture; c) performing nucleotide amplification reaction of the amplification mixture and quantifying the amplification products; d) analysing the results of the quantification of the amplification products, thereby determining the status of the differentiated iPSCs.
  • the differentiated iPSCs-derived sample provided in step a) may be a sample prepared from differentiated iPSCs.
  • the method may comprise as a first step the additional step 0) of preparing samples from differentiated iPSCs, thereby obtaining a differentiated iPSCs-derived sample.
  • This differentiated iPSCs-derived sample of optional step 0) is the differentiated iPSCs-derived sample provided in step a).
  • the differentiated iPSCs-derived sample provided in step a) may be total RIMA isolated from the differentiated iPSCs or molecules derived from total RNA isolated from the differentiated iPSCs (e.g., cDNA).
  • the differentiated iPSCs- derived sample provided in step a) is preferably a cDNA sample, preferably obtained by reverse transcription reaction from RIMA isolated from the differentiated iPSCs.
  • step d) of the method the results of the quantification of the amplification products are analysed and thereby, the status of the differentiated iPSCs is determined.
  • the differentiated iPSCs-derived sample aliquot which was mixed with an amplification primer set capable of amplifying a gene specific for endodermal cells or a part thereof yields a positive result (indicating that the sample contains or is derived from endodermal cells)
  • the analysed sample of the differentiated iPSCs comprises endodermal cells.
  • the result is negative, it can be concluded that the analysed sample of the differentiated iPSCs does not comprise endodermal cells.
  • the analysis allows to obtain a score, which ranges from 0 to 1.
  • a score from 0 to 0.5 would indicate that the differentiated iPSCs do not have the expected/desired status.
  • the score is obtained by multiplying the quality parameter obtained in the analysis with its certainty of classification p.
  • a score from more than 0.5 to 1 would indicate that the differentiated iPSCs have achieved the expected/desired status.
  • This in vitro method for determining the status of differentiated iPSCs preferably comprises at least one of the following steps: b2) mixing a second aliquot of the differentiated iPSCs-derived sample with a second set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture; b3) mixing a third aliquot of the differentiated iPSCs-derived sample with a third set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture.
  • the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof is a gene specific for the same differentiation state as the differentiation state selected in step bl) of the in vitro method for determining the status of differentiated iPSCs.
  • the method may comprise one of the additional steps b2) and b3), or both of the additional steps b2) and b3).
  • the order of the steps bl), b2), and b3) is not fixed. It is also possible that all steps bl), b2), and/or b3) are performed at the same time. The only requirement is that the steps bl), b2), and b3) are performed after step a) and before step c).
  • an in vitro method for determining the status, especially the differentiation status, of iPSCs comprising the following steps: a) providing an iPSCs-derived sample; bla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2a) mixing a second aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3a) mixing a third aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4a) mixing a fourth ali
  • the method for determining the status of iPSCs of the present invention may be used to monitor the status of iPSCs obtained by reprogramming somatic cells during cultivation of said iPSCs in cell culture.
  • the status of the iPSCs may be the differentiation status of the iPSCs, which indicates whether a sample taken from an iPSCs cell culture is of the undifferentiated state (pluripotent), or of any of the three primary germ layers (endoderm, ectoderm, mesoderm), mixtures thereof, or none of the aforementioned states, e.g., is a further differentiated cell sample or a cell sample of non-iPSC origin.
  • iPSCs obtained by reprogramming somatic cells may, e.g., be used for patient-specific disease modelling or for treatment of patients with their own cells which have been treated ex vivo.
  • the cells are only grown under controlled conditions (cell culture) and subcultured or passaged to prolong the lifespan and/or to increase the numbers of cells in culture whenever necessary.
  • new culture medium must be added at regular intervals, e.g., to maintain the pluripotency of the cells.
  • the iPSCs may lose their pluripotency, i.e., they may lose their potential to differentiate into any of the three primary germ layers endoderm, ectoderm, and mesoderm.
  • the iPSCs are not any more suitable to be used for patient-specific disease modelling or for treatment of patients with their own cells which have been treated ex vivo.
  • the iPSCs-derived sample provided in step a) may be prepared from cells taken from an iPSCs cell culture grown under controlled conditions, e.g., an ordinary or normal iPSCs cell culture.
  • the cells used to prepare the iPSCs- derived sample may be taken from said cell culture directly before or after passaging, or after a longer cultivation interval in which the cells did not receive new culture medium (e.g., a weekend).
  • the method may comprise as a first step the additional step 0) of preparing samples from an iPSCs cell culture, thereby obtaining an iPSCs-derived sample.
  • This iPSCs-derived sample of optional step 0) is the iPSCs-derived sample provided in step a).
  • the iPSCs- derived sample provided in step a) may also be derived from other cells, provided that the said cells originate from iPSCs.
  • the iPSCs-derived sample provided in step a) may be total RIMA isolated from the cells taken from the iPSCs cell culture or molecules derived from total RNA isolated from cells taken from the iPSCs cell culture (e.g., cDNA).
  • the iPSCs- derived sample provided in step a) is preferably a cDNA sample, preferably obtained by reverse transcription reaction from RNA isolated from the cells taken from the iPSCs cell culture.
  • step b2a) is performed before step bla
  • step b3a) is performed before step bla
  • all steps bla), b2a), b3a), and b4a) are performed at the same time.
  • the only requirement is that the steps bla), b2a), b3a), and b4a) are performed after step a) and before step c) of the method of the invention. It is also possible that only one of the steps bla), b2a), b3a), and b4a) is performed in the method of the invention after step a) and before step c).
  • step d) of the method the results of the quantification of the amplification products are analysed and thereby, the status of the iPSCs is determined.
  • the iPSCs-derived sample aliquot which was mixed with the amplification primer set capable of amplifying a gene specific for pluripotent cells or a part thereof yields a positive result (indicating that the sample contains or is derived from pluripotent cells)
  • the analysed sample of the iPSCs comprises pluripotent cells. If the result is negative, it can be concluded that the analysed sample of the iPSCs does not comprise pluripotent cells.
  • the analysis allows to obtain a score, which in the case that only one of the steps bla), b2a), b3a), and b4a) was performed ranges from 0 to 1.
  • a score from 0 to 0.5 would then indicate that the cells do not have the expected/desired status.
  • a score from more than 0.5 to 1 would indicate that the cells have achieved the expected/desired status.
  • the total score would be from 0 to 4, because each of the four subtests (pluripotent, endoderm, ectoderm, mesoderm) would lead to a score between 0 and 1, and the respective subtest scores are summed up.
  • the respective subtest scores are obtained by multiplying the quality parameter obtained in each subtest with its certainty of classification p.
  • This in vitro method for determining the status of iPSCs preferably additionally comprises at least one of the following steps: bib) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of
  • the method of the invention may comprise one of the additional steps bib), b2b), b3b), and b4b), two of the additional steps bib), b2b), b3b), and b4b), three of the additional steps bib), b2b), b3b), and b4b), or four of the additional steps bib), b2b), b3b), and b4b).
  • step bib is performed after step bla) or before step bla. It is also possible that step bib) is performed after step b4a), and so on. It is also possible that all steps bla), b2a), b3a), b4a), bib), b2b), b3b), and b4b) are performed at the same time.
  • This in vitro method for determining the status of iPSCs preferably additionally comprises at least one of the following steps: blc) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers
  • the method of the invention may comprise one of the additional steps blc), b2c), b3c), and b4c), two of the additional steps blc), b2c), b3c), and b4c), three of the additional steps blc), b2c), b3c), and b4c), or four of the additional steps blc), b2c), b3c), and b4c).
  • step blc is performed after step bla), before step bla), after step bib), or before step bib. It is also possible that step blc) is performed after step b4a) or after step b4b), and so on. It is also possible that all steps bla), b2a), b3a), b4a), bib), b2b), b3b), b4b), blc), b2c), b3c), and b4c) are performed at the same time.
  • step blc), b2c), b3c), and b4c) - like the steps bla), b2a), b3a), b4a), bib), b2b), b3b), and b4b) - are performed after step a) and before step c) of the method of the invention.
  • a computer- implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample comprising the following steps: providing an iPSC-derived sample to be analysed; performing a PCR analysis on the iPSC-derived sample and obtaining fluorescence signal-derived values for n cycles of the PCR.; determining a quality parameter for the iPSC-derived sample using a trained classifier and the obtained fluorescence signal- derived value.
  • iPSC induced pluripotent stem cell
  • the fluorescence signal- derived value is used as an input for the classifier.
  • the fluorescence signal- derived value may comprise an intensity value for each cycle of the PCR.
  • the classifier may be based on a Support Vector Machine (SVM), Random Forest or an Artificial Neural Network (ANN).
  • the quality parameter may be a binary parameter having values of 0 (e.g., indicating that the sample does not contain or is not derived from pluripotent cells, or indicating that the sample does not contain or is not derived from endodermal cells) and 1 (e.g., indicating that the sample contains or is derived from pluripotent cells, or indicating that the sample contains or is derived from endodermal cells).
  • the trained classifier may be trained with training data comprising pairs of fluorescence signal-derived values and classification data corresponding to each fluorescence signal-derived value and characterising the integrity of an iPSC-derived sample.
  • the quality of the iPSC-derived sample may allow a statement about the pluripotency status of the iPSCs and/or the differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm. Quality parameters of each of the differentiation states can be used to subsequently determine a score indicating the integrity of the iPSCs. This score is called "hiPSCore". To calculate the hiPSCore, the quality parameter of each of the differentiation states (subtests) is multiplied with its certainty of classification p, and the resulting products are summed up, yielding the hiPSCore (see Fig. 1 A).
  • the quality parameters can be used to subsequently determine the status of differentiated iPSCs (in the in vitro method for determining the status of differentiated iPSCs of the present invention), or to subsequently determine the status, especially the differentiation status, of iPSCs (in the in vitro method for determining the status, especially the differentiation status, of iPSCs of the present invention).
  • the PCR analysis performed on the iPSC-derived sample may be a quantitative polymerase chain reaction (qPCR) analysis or a digital polymerase chain reaction (dPCR) analysis.
  • qPCR quantitative polymerase chain reaction
  • dPCR digital polymerase chain reaction
  • a qPCR analysis is performed, and the fluorescence signal-derived values obtained for n cycles of the PCR are the cycle threshold (Ct) values, i.e., the cycle at which the signal intensity significantly crosses the background signal, obtained by the qPCR device.
  • Ct cycle threshold
  • the rating may define the accuracy of the classifier or e.g., the speed of the classification process.
  • different classifiers can be tested, and the best classifier can be used for the classification of the fluorescence signal-derived value.
  • the different classifiers tested may be e.g., an ANN, a CNN and a SVM.
  • the different classifiers can be of the same type (such as an SVM), while the parameters of the classifier are varied in order to obtain the optimum classifier, pl may be 70 and p2 may be 30.
  • RNA isolation, cDNA transcription and qPCR were performed by employing two different commercially available kits, namely the StemMACS Trilineage Differentiation kit (Miltenyi Biotec) and the StemDIFF Trilineage Differentiation kit (STEMCELL Technologies) according to the manufacturers' instructions. Accordingly, 5 * 10 ⁇ 4 (mesoderm)/2 * 10 ⁇ 5 (endoderm, ectoderm) iPSCs were seeded for differentiation in Geltrex-coated wells of a 24-well plate. After five (STEMCELL: endoderm, mesoderm) and seven days (rest) of differentiation, the cells were washed twice with PBS and harvested into 300 pl Trizol (Thermo). RNA isolation, cDNA transcription and qPCR
  • RNA isolation was performed using the Direct-zol RNA Miniprep kit (Zymo) including on-column DNAse digestion according to the manufacturers' instructions.
  • RNA was eluted in 25 pl aqua dest. and concentration measured on a NanoDrop2000 spectrophotometer (Thermo). 1 pg of RNA was reverse transcribed using the HiScript III RT SuperMix for qPCR kit (Vazyme). Afterwards, cDNA was diluted 1/10 with aqua dest.
  • RNA of two biological replicates of iPSC pluripotent control cells and cells differentiated with the StemMACS Trilineage differentiation kit were used for first strand cDNA synthesis in a 6 pl reaction consisting of 2 pl 10 pM RT primer (5'->3': AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TV; SEQ ID NO: 25; The "V” in the primer sequence stands for "not T", so can be any DNA base other than T (A, C, G). This primer is a degenerated primer to prevent poly(A) slippage.
  • Poly(A) slippage is caused by the mRNA poly(A) tail which can cause mistakes during reverse transcription. This is prevented by degenerate primers adding a "non-T" to the primer binding to poly(A).), 1 pl dNTPs (10 mM each, Vazyme) and aqua dest. for 5 min at 70 °C.
  • Second strand synthesis was performed by adding 2.5 pl Template Switching Buffer (New England Biolabs), 0.5 pl 75 pM Template Switching Oligo (5'->3': GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG; SEQ ID NO: 26; The "rG” in the primer sequence stand for the RNA base guanine.) and 1 pl Template Switching RT Enzyme Mix (New England Biolabs) to the first strand reaction and incubating 90 min at 42 °C, followed by heat inactivation at 85 °C for five minutes.
  • 2.5 pl Template Switching Buffer New England Biolabs
  • 0.5 pl 75 pM Template Switching Oligo 5'->3': GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG; SEQ ID NO: 26; The "rG” in the primer sequence stand for the RNA base guanine.
  • Library preparation was performed according to the Ligation sequencing amplicons - Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies, SQK- NBD114.24) according to the version NBA_9168_vl l4_revE_15Sep2022. Accordingly, equal amounts of double-stranded cDNA (20.25 ng) were subjected to library preparation. The final library was quantified on a Qubit Fluorometer (Thermo) using the dsDNA HS kit (Thermo). Library size was determined using a D1000 Screen Tape assay (Agilent). A final amount of 15 fmol was loaded on a P2 solo flow cell and sequenced for 72 h.
  • Differentially expressed transcripts were assessed by normalizing Iog2 counts to the pluripotent samples and filtering for high Iog2 fold changes (I2fc, depending on the differentiation state higher than 3 to 6 for differentiated samples and lower than -2 for each differentiated sample vs the pluripotent controls). All data was visualized using ggplot2 (v.3.4.2) and ComplexHeatmap (v.2.14).
  • iPSC induced pluripotent stem cell

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Abstract

The present invention relates to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs). The present invention further relates to a kit for determining the integrity of iPSCs, to an in vitro method for determining the status of differentiated iPSCs, to an in vitro method for determining the status of iPSCs, especially the differentiation status of iPSCs, and to a computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample.

Description

Method for the determination of pluripotency of stem cells
The present invention relates to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs). The present invention further relates to a kit for determining the integrity of iPSCs, to an in vitro method for determining the status of differentiated iPSCs, to an in vitro method for determining the status of iPSCs, especially the differentiation status of iPSCs, and to a computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample.
Induced pluripotent stem cells (iPSCs), especially human induced pluripotent stem cells (hiPSCs), are a versatile tool to study physiological processes in a meaningful way. Human induced pluripotent stem cells have revolutionized basic and translational research as well as regenerative medicine. These cells are derived from somatic cells which have been reprogrammed with pluripotency factors yielding cells with stem cell-like features including capability to differentiate into cells of each of the three primary germ layers, endoderm, mesoderm, and ectoderm (Takahashi, K.; Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 2006, 126, 663-676, doi: 10.1016/j.cell.2006.07.024; Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 2007, 131, 861-872, doi : 10.1016/j. cell.2007.11.019; Moradi, S.; Mahdizadeh, H.; Saric, T.; Kim, J.; Harati, J.; Shahsavarani, H.; Greber, B.; Moore, J.B. Research and Therapy with Induced Pluripotent Stem Cells (IPSCs): Social, Legal, and Ethical Considerations. Stem Cell Res Ther 2019, 10, 341, doi : 10.1186/sl3287-019-1455-y). Human iPSCs can be genetically modified, e.g., by CRISPR/Cas, which further increases their use range application and research spectrum (Hockemeyer, D.; Jaenisch, R. Induced Pluripotent Stem Cells Meet Genome Editing. Cell Stem Cell 2016, 18, 573-586). They not only enable patient-specific disease modelling, e.g., by organoid generation to model development and pathophysiology of organs, but more importantly enable treatment of patients with their own cells which have been treated ex vivo (Scudellari, M. How IPS Cells Changed the World. Nature 2016, 534, 310-312, doi : 10.1038/534310a; Hofer, M.; Lutolf, M.P. Engineering Organoids. Nat Rev Mater 2021, 6, 402-420; Kim, J.; Koo, B.K.; Knoblich, J. A. Human Organoids: Model Systems for Human Biology and Medicine. Nat Rev Mol Cell Biol 2020, 21, 571-584).
These points make it very clear that, in order to guarantee coherence of research, standardisation, and patient safety, iPSC lines in use need to be quality-checked to ensure their integrity. Such quality control includes assessment of genome (both nuclear and mitochondrial) integrity, as well as assessment of pluripotency status and differentiation capability (Rossi, A.; Lickfett, S.; Martins, S.; Prigione, A. A Call for Consensus Guidelines on Monitoring the Integrity of Nuclear and Mitochondrial Genomes in Human Pluripotent Stem Cells. Stem Cell Reports 2022, 17, 707-710; Allison, T.F.; Andrews, P.W.; Avior, Y.; Barbaric, I.; Benvenisty, N.; Bock, C.; Brehm, J.; Brustle, O.; Damjanov, I.; Elefanty, A.; et al. Assessment of Established Techniques to Determine Developmental and Malignant Potential of Human Pluripotent Stem Cells. Nat Commun 2018, 9, doi : 10.1038/s41467-018-04011- 3).
A variety of possible tests exist, but analysis markers are often only suggested, complicating comparability of even commercially used cell lines across laboratories (Allison, T.F.; Andrews, P.W.; Avior, Y.; Barbaric, I.; Benvenisty, N.; Bock, C.; Brehm, J.; Brustle, 0.; Damjanov, I.; Elefanty, A.; et al. Assessment of Established Techniques to Determine Developmental and Malignant Potential of Human Pluripotent Stem Cells. Nat Commun 2018, 9, doi : 10.1038/s41467-018-04011-3; Sullivan, S.; Stacey, G.N.; Akazawa, C.; Aoyama, N.; Baptista, R.; Bedford, P.; Bennaceur Griscelli, A.; Chandra, A.; Elwood, N.; Girard, M.; et al. Quality Control Guidelines for Clinical-Grade Human Induced Pluripotent Stem Cell Lines. Regenerative Med 2018, 13, 859- 866, doi: 10.2217/rme-2018-0095; Kuang, Y.-L.; Munoz, A.; Nalula, G.; Santostefano, K.E.; Sanghez, V.; Sanchez, G.; Terada, N.; Mattis, A.N.; lacovino, M.; Iribarren, C.; et al. Evaluation of Commonly Used Ectoderm Markers in IPSC Trilineage Differentiation. Stem Cell Res 2019, 37, 101434, doi : 10.1016/j.scr.2019.101434).
The most frequently used test to assess differentiation capability is the formation and spontaneous differentiation of embryoid bodies (EBs) which is still based on a protocol first described in the 1970s (Martin, G.R.; Evans, M . Differentiation of Clonal Lines of Teratocarcinoma Cells: Formation of Embryoid Bodies In Vitro (Mouse Tumors/Tissue Culture/Pluripotent Cells/Cell Determination/Endoderm); 1975; Vol. 72). The major disadvantage of this approach lies within its spontaneous stochastic nature, yielding different ratios of differentiated cells for each experimental replicate, complicating comparability and detection of germ layer-specific targets for the expectable low differentiation efficiencies. Detection is usually performed employing immunofluorescent (IF) imaging or fluorescence-activated cell sorting (FACS). Although these methods enable detection on a single cell scale, they require a substantial amount of hands-on time, careful selection of markers, timing of analysis, or are not quantifiable (IF). In addition, standardization cannot be achieved as they are mostly assessed manually.
Quantitative real time PCR (RT-qPCR) can be used to detect relative gene expression with high sensitivity. Although RT-qPCR is a standard method in molecular biology, commercially available RT-qPCR-based solutions are expensive, are only tailored to spontaneous differentiation of EBs, and are not developed over time. One example is the Scorecard approach which was developed to enable detection of germ layer-specific marker gene expressions of EBs (Tsankov, A.M.; Akopian, V.; Pop, R.; Chetty, S.; Gifford, C.A.; Daheron, L.; Tsankova, N.M.; Meissner, A. A QPCR ScoreCard Quantifies the Differentiation Potential of Human Pluripotent Stem Cells. Nat Biotechnol 2015, 33, 1182-1192, doi: 10.1038/nbt.3387). While this offers at least some measure of streamlining, it is very expensive, posing a major financial issue for labs frequently performing iPSC quality control. Its significance is also debated as there is currently no predictive power underlying these tests and any cell line is likely to pass the test.
Another example is Sekine, K. et al., Robust detection of undifferentiated iPSC among differentiated cells. Scientific Reports 2020, 10, 10293, disclosing that marker genes, which are expressed specifically and highly in undifferentiated iPSC, were selected from single cell RNA sequence data to perform detections of residual undifferentiated cells in differentiated cell products.
Another option to assess differentiation capability is the teratoma assay which is based on spontaneous tumour formation of transplanted iPSCs into immunocompromised mice in vivo (Wesselschmidt, R.L. The Teratoma Assay: An in Vivo Assessment of Pluripotency. Methods Mol Biol 2011, 767, 231-241, doi : 10.1007/978-l-61779-201-4_17). Handling mice, however, is cost- and administration-extensive and such tests employed as a mere quality check unnecessarily involve animal harm. In addition, teratoma formation suffers from the same stochastic nature as in vitro differentiation of EBs.
While many markers indicating pluripotency/differentiation status are known, they mostly have been identified using next generation sequencing (NGS) based on short reads and/or microarrays. While NGS is a powerful technique, some transcripts might not be detected. In fact, the current uprising of long read sequencing has shown that indeed different transcripts are detected when comparing the two methods. Reassessment of pluripotency/differentiation markers is therefore paramount, especially considering potential overlaps of gene expression patterns, such as e.g., S0X2 which is still used as a pluripotency marker but also highly expressed in ectoderm differentiation (Zhang, S. Sox2, a Key Factor in the Regulation of Pluripotency and Neural Differentiation. World J Stem Cells 2014, 6, 305, doi : 10.4252/wjsc.v6.i3.305).
As can be seen from the above, there exists a need for in vitro methods for determining the integrity of iPSCs.
The object of the present invention is therefore to provide an in vitro method for determining the integrity of iPSCs, a kit for determining the integrity of iPSCs, in vitro methods for determining the status of iPSCs or differentiated iPSCs, and a computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample which are able to overcome at least some of the disadvantages of the prior art.
The object of the present invention is solved by an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs) comprising the following steps: a) differentiating samples of the iPSCs into endodermal, ectodermal, and mesodermal cells; b) preparing samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells; cla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2a) mixing a first aliquot of the endodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3a) mixing a first aliquot of the ectodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4a) mixing a first aliquot of the mesodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; d) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; e) analysing the results of the quantification of the amplification products, thereby obtaining a score that allows to assess the integrity of the iPSCs.
Surprisingly it was found that the method of the present invention allows the determination of the integrity of iPSCs in a convenient, fast, easy-to-use, easy- to-access, and easily quantifiable way, enables unequivocal characterisation of analysed samples, and provides means to screen many samples simultaneously.
The method for determining the integrity of iPSCs of the present invention is an in vitro method. The integrity of the iPSCs determined in the method of the invention may be the pluripotency status of the iPSCs and/or the differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm. The pluripotency status indicates whether the iPSCs are pluripotent, i.e., whether they theoretically have the potential to differentiate into any of the three primary germ layers: endoderm (progenitor of gut, lungs, yolk sac), ectoderm (progenitor of nervous, sensory, epidermis), and mesoderm (progenitor of muscle, skeleton, blood vascular, urogenital, dermis). The differentiation capacity indicates whether the iPSCs actually differentiate into the three primary germ layers endoderm, ectoderm, and mesoderm. In a preferred embodiment, the integrity of the iPSCs is therefore the pluripotency status of the iPSCs and/or the differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm. The method of the present invention can be used to determine the quality of iPSCs obtained by reprogramming somatic cells and which may, e.g., be used for patient-specific disease modelling or for treatment of patients with their own cells which have been treated ex vivo. The method of the present invention can also be used to determine the quality of iPSCs which have been manipulated, e.g., by genome editing. For this, a sample of said iPSCs may be analysed with the method of the present invention. Conclusions about the quality of the iPSCs from which the sample was taken can be drawn from the analysis results.
In step a) of the method for determining the integrity of iPSCs according to the present invention, some of the iPSCs, i.e., pluripotent stem cells generated directly from a somatic cell, which may also be iPSCs manipulated by genome editing, to be evaluated are differentiated into endodermal, ectodermal, and mesodermal cells. I.e., the method of the present invention may comprise the following step a): differentiating the iPSCs into endodermal, ectodermal, and mesodermal cells. Methods for differentiating samples of iPSCs into endodermal, ectodermal, and mesodermal cells are known to the person skilled in the art.
In step b) of the method for determining the integrity of iPSCs according to the present invention, samples are prepared of the iPSCs which were not differentiated, and from the endodermal, ectodermal, and mesodermal cells generated in step a) by differentiating the iPSCs. Accordingly, in step b), samples are prepared from four different cell types, i.e., from pluripotent cells (iPSCs), from endodermal cells, from ectodermal cells, and from mesodermal cells.
In the next steps of the method for determining the integrity of iPSCs according to the present invention, a first aliquot of each of the samples prepared in step b) is mixed with a first set of amplification primers capable of amplifying a gene specific for the respective cell type, or a part of said gene specific for the respective cell type. As described previously with respect to step b), samples from four different cell types (pluripotent cells/ iPSCs, endodermal cells, ectodermal cells, mesodermal cells) are prepared in the method of the invention. Therefore, the method of the invention requires at least four mixing steps, which are designated as steps cla), c2a), c3a), and c4a). In step cla), a first aliquot of the iPSCs-derived sample (i.e., the pluripotent cells-derived sample) is mixed with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part of a gene specific for pluripotent cells, thereby forming an amplification mixture cla). In step c2a), a first aliquot of the endodermal cells-derived sample is mixed with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part of a gene specific for endodermal cells, thereby forming an amplification mixture. In step c3a), a first aliquot of the ectodermal cells-derived sample is mixed with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part of a gene specific for ectodermal cells, thereby forming an amplification mixture. In step c4a), a first aliquot of the mesodermal cells- derived sample is mixed with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part of a gene specific for mesodermal cells, thereby forming an amplification mixture.
A gene specific for pluripotent cells is a gene that is only expressed at high levels in pluripotent cells, but not at high levels in endodermal, ectodermal, and mesodermal cells. Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in pluripotent cells, but not at high levels in endodermal, ectodermal, and mesodermal cells, respectively. Therefore, such a gene may be used to unequivocally determine the differentiation state "pluripotent" (by analysing levels of mRNA molecules). A part of the gene specific for pluripotent cells may, e.g., be a fragment or segment of the gene specific for pluripotent cells. It may be advantageous to use amplification primers capable of amplifying such part of a gene specific for pluripotent cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for pluripotent cells, e.g., by amplifying cDNA.
A gene specific for endodermal cells is a gene that is only expressed at high levels in endodermal cells, but not at high levels in pluripotent, ectodermal, and mesodermal cells. Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in endodermal cells, but not at high levels in pluripotent, ectodermal, and mesodermal cells, respectively. Therefore, such gene may be used to unequivocally determine the differentiation state "endodermal" (by analysing levels of mRNA molecules). A part of the gene specific for endodermal cells may, e.g., be a fragment or segment of the gene transcript specific for endodermal cells. It may be advantageous to use amplification primers capable of amplifying such part of a gene specific for endodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for endodermal cells, e.g., by amplifying cDNA.
A gene specific for ectodermal cells is a gene that is only expressed at high levels in ectodermal cells, but not at high levels in pluripotent, endodermal, and mesodermal cells. Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in ectodermal cells, but not at high levels in pluripotent, endodermal, and mesodermal cells, respectively. Therefore, such gene may be used to unequivocally determine the differentiation state "ectodermal" (by analysing levels of mRNA molecules). A part of the gene specific for ectodermal cells may, e.g., be a fragment or segment of the gene specific for ectodermal cells. It may be advantageous to use amplification primers capable of amplifying such part of a gene specific for ectodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for ectodermal cells, e.g., by amplifying cDNA.
A gene specific for mesodermal cells is a gene that is only expressed at high levels in mesodermal cells, but not at high levels in pluripotent, endodermal, and ectodermal cells. Gene expression products, i.e., transcribed and spliced mRNA, of the said gene are thus present at high levels only in mesodermal cells, but not at high levels in pluripotent, endodermal, and ectodermal cells, respectively. Therefore, such gene may be used to unequivocally determine the differentiation state "mesodermal" (by analysing levels of mRNA molecules). A part of the gene specific for mesodermal cells may, e.g., be a fragment or segment of the gene specific for mesodermal cells. It may be advantageous to use amplification primers capable of amplifying such part of a gene specific for mesodermal cells, because the length of such part of a gene to be amplified (number of nucleotides to be amplified) can be individually defined. It is to be understood that in the method of the invention, the amplification primers may be used to amplify the gene expression products or a part thereof of the respective gene specific for mesodermal cells, e.g., by amplifying cDNA.
The order of the steps cla), c2a), c3a), and c4a) in the method of the invention is not fixed. It is possible that step c2a) is performed before step cla), that step c3a) is performed before step cla), and so on. It is also possible that all steps cla), c2a), c3a), and c4a) are performed at the same time. The only requirement is that the steps cla), c2a), c3a), and c4a) are performed after step b) and before step d) of the method of the invention.
In step d) of the method for determining the integrity of iPSCs according to the present invention, the amplification mixtures generated in steps cla), c2a), c3a), and c4a) are processed further. The at least four amplification mixtures obtained in the method of the invention are subjected to amplification reactions and the resulting amplification products are quantified. It is to be understood that for quantifying the respective amplification products, the amount of the amplification products may be normalised relative to the amount of at least one reference gene, i.e., a gene that is expressed in the target cells (iPSCs, endodermal, ectodermal, and mesodermal cells) at a similar level. Suitable reference genes, such as GAPDH, ACTB, PPIA, etc., are known to the person skilled in the art.
In step e) of the method for determining the integrity of iPSCs according to the present invention, the results of the quantification of the amplification products are analysed, e.g., by using a software implemented on a computer, thereby obtaining a score that allows to assess the integrity of the iPSCs. This final score indicating the integrity of the iPSCs is also called "hiPSCore" in the following.
In the method of the invention, at least one gene specific for iPSCs or part thereof, at least one gene specific for endodermal cells or part thereof, at least one gene specific for ectodermal cells or part thereof, and at least one gene specific for mesodermal cells or part thereof is amplified, quantified, and analysed. The quality of the analysis result obtained in step e) of the method of the invention, i.e., the quality of the score obtained in step e) of the method of the invention, may be improved by amplifying, quantifying, and analysing more than one gene specific for iPSCs, endodermal cells, ectodermal cells, or mesodermal cells, respectively.
In a preferred embodiment of the invention, the method therefore additionally comprises at least one of the following steps: clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2b) mixing a second aliquot of the endodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3b) mixing a second aliquot of the ectodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4b) mixing a second aliquot of the mesodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
The method of the invention may comprise one of the additional steps clb), c2b), c3b), and c4b), two of the additional steps clb), c2b), c3b), and c4b), three of the additional steps clb), c2b), c3b), and c4b), or all of the four of the additional steps clb), c2b), c3b), and c4b).
The order of the additional steps clb), c2b), c3b), and c4b) in the method of the invention is not fixed. It is, e.g., possible that additional step clb) is performed after step cla) or before step cla). It is also possible that step clb) is performed after step c4a), and so on. It is also possible that all steps cla), c2a), c3a), c4a), clb), c2b), c3b), and c4b) are performed at the same time. The only requirement is that the additional steps clb), c2b), c3b), and c4b) - like the steps cla), c2a), c3a), and c4a) - are performed after step b) and before step d) of the method of the invention.
The quality of the analysis result obtained in step e) of the method of the invention, i.e., the quality of the score obtained in step e) of the method of the invention, may be even further improved by amplifying, quantifying, and analysing more than two genes specific for iPSCs, endodermal cells, ectodermal cells, or mesodermal cells. In a further preferred embodiment of the invention, the method therefore additionally comprises at least one of the following steps: clc) mixing a third aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2c) mixing a third aliquot of the endodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3c) mixing a third aliquot of the ectodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4c) mixing a third aliquot of the mesodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
The method of the invention may comprise one of the additional steps clc), c2c), c3c), and c4c), two of the additional steps clc), c2c), c3c), and c4c), three of the additional steps clc), c2c), c3c), and c4c), or all of the four of the additional steps clc), c2c), c3c), and c4c).
The order of the additional steps clc), c2c), c3c), and c4c) in the method of the invention is not fixed. It is, e.g., possible that additional step clc) is performed after step cla), before step cla), after step clb), or before step clb). It is also possible that step clc) is performed after step c4a) or after step c4b), and so on. It is also possible that all steps cla), c2a), c3a), c4a), clb), c2b), c3b), c4b), clc), c2c), c3c), and c4c) are performed at the same time. The only requirement is that the additional steps clc), c2c), c3c), and c4c) - like the steps cla), c2a), c3a), c4a), clb), c2b), c3b), and c4b) - are performed after step b) and before step d) of the method of the invention. Thus, in a preferred embodiment, the present invention refers to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs) comprising the following steps: a) differentiating samples of the iPSCs into endodermal, ectodermal, and mesodermal cells; b) preparing samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells; cla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2a) mixing a first aliquot of the endodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c2b) mixing a second aliquot of the endodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3a) mixing a first aliquot of the ectodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c3b) mixing a second aliquot of the ectodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4a) mixing a first aliquot of the mesodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; c4b) mixing a second aliquot of the mesodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; d) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; e) analysing the results of the quantification of the amplification products, thereby obtaining a score that allows to assess the integrity of the iPSCs.
In a further preferred embodiment, the present invention thus refers to an in vitro method for determining the integrity of induced pluripotent stem cells (iPSCs) comprising the following steps: a) differentiating samples of the iPSCs into endodermal, ectodermal, and mesodermal cells; b) preparing samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells; cla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; clc) mixing a third aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2a) mixing a first aliquot of the endodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c2b) mixing a second aliquot of the endodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c2c) mixing a third aliquot of the endodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3a) mixing a first aliquot of the ectodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c3b) mixing a second aliquot of the ectodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c3c) mixing a third aliquot of the ectodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4a) mixing a first aliquot of the mesodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; c4b) mixing a second aliquot of the mesodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; c4c) mixing a third aliquot of the mesodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; d) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; e) analysing the results of the quantification of the amplification products, thereby obtaining a score that allows to assess the integrity of the iPSCs.
The method of the present application can be applied to any induced pluripotent stem cells (iPSCs). In a preferred embodiment, the iPSCs are human induced pluripotent stem cells (hiPSCs), as these are a versatile tool to study physiological processes in a meaningful way.
The in vitro method of the present invention comprises amplification of genes specific for pluripotent cells, amplification of genes specific for endodermal cells, amplification of genes specific for ectodermal cells, and amplification of genes specific for mesodermal cells, or amplification of parts of the aforementioned genes. The following genes (or parts thereof) are preferably amplified and analysed according to the present invention :
(i) the gene specific for pluripotent cells may be at least one selected from the group consisting of CNMD, NANOG, SPP1, DHRS3, L1TD1, TMEM125, SYT14, SMPDL3B, LCK, SF3B4, PDPN, RAB42, VASH2, RABGAP1L, VENT5B, VSNL1, ADD2, AP1S3, B3GNT7, UGP2, CRYGD, TUBA4A, KHK, CHAC2, CDCP1, GPD1L, TIMP4, IFRD2, CAMKV, DPPA4, SYNP02, ZFP42, RASL11B, APELA, TRIML2, NSG1, CXCL5, KLKB1, SCGB3A2, POLR3G, N0P16, HRH2, PAK1IP1, PSMB8, PSMB9, P0U5F1, TBRG4, MACC1, MAGI2, RARRES2, CCL26, DIAPH2, PIM2, ZMAT4, PRDM14, FABP5, RRS1, B0P1, TERFI, IDO1, ENSG00000258417, HHLA1, COL14A1, P0U5F1B, E2F8, ZNF483, ZNF215, GRIA4, FAM111B, FOSL1, KCTD14, GLB1L3, FGF19, NRIP3, CUZD1, CCDC172, HTR7, ZNF485, TEAD4, SLC38A4, PHC1, METTL1, SLC6A15, MGAT4C, HSPH1, EDNRB, CCDC169, DCLK1, MTHFD1, EGLN3, GPR176, RBPMS2, AEN, CKMT1B, CKMT1A, MT1X, NAN0GP8, SCG3, GDPGP1, VAT1L, DDX28, CHST4, ALG1, SYNGR3, C1QBP, PIGW, PPP1R1B, PIPOX, RBF0X3, HMSD, FERMT1, TNNC2, DNMT3B, FXYD7, CYP2S1, TOMM40, POLR1G, PPM IN, SYN3, and MCM5, preferably at least one selected from the group consisting of CNMD, NANOG, and SPP1 ; and/or
(ii) the gene specific for endodermal cells may be at least one selected from the group consisting of GATA6, EOMES, CER1, F3, PIFO, AGL, PDZK1, CD48, DUSP10, ITLN2, KIAA0040, USH2A, FYB2, KCNK1, RAB25, AP0A2, SLC5A9, MIXL1, LEFTY1, LEFTY2, FMN2, S100A16, S100A14, ELF3, CAPN13, LRP1B, KCNJ3, STAT4, PELI1, KCNK12, TTN, CCDC141, RAB17, CLIP4, VIL1, VAMP8, C0BLL1, CD80, UPK1B, TAGLN3, CLDN18, EHHADH, CLDN11, PLSCR1, RARB, TRMT44, ETN PPL, TMPRSS11E, PPM IK, AADAT, AC0X3, MFAP3L, CPE, ACSL1, ANXA3, GPM6A, RASSF6, PLCXD3, FST, CLVS2, TRDN, GMPR, SAMD3, GMPR, EGFL8, MANEA, HLA-DQB1, VEGFA, SEMA3E, CALCR, KIAA0895, EPHB6, KEL, STEAP1B, CAST0R2, PRSS1, PRSS2, PLXNA4, GRPR, NR0B1, KLF8, CLTRN, PORCN, ARSL, STC1, KIF13B, TRPA1, LY6E, FAM135B, CSMD3, FGF17, PIP5K1B, CTSV, TTC39B, GALNT12, PTGDS, D0CK8, CCKBR, DSCAML1, CST6, 0R52A1, 0VCH2, GRAM DIB, MY03A, CPY26A1, PLAC9, IGFBP6, TESPA1, CNNM1, SLC2A3, NTN4, NUDT4, LGR5, SLC2A14, LMNTD1, ACSM4, DNAJC15, KLF5, F10, EPSTI1, GSC, 0TX2, UNC13C, PIEZ01, YPEL3, PMFBP1, HP, ARL4D, NTN1, MFAP4, TVP23C, PIK3R5, KRT19, CELF4, RNF152, POTEC, F0XA2, WFD2C, CST1, DBNDD2, HNF4A, TLE2, NCR1, NLRP7, CD70, TNFSF14, HPN, APOE, SULT2B1, GIPR, APOCI, C3, RIPPLY3, DSCAM, and ERVH48-1, preferably at least one selected from the group consisting of GATA6, EOMES, and CER1 ,- and/or
(iii) the gene specific for ectodermal cells may be at least one selected from the group consisting of PAX6, HESS, PAMR1, DLK1, P0U3F1, ATP1A2, SYT11, DPYSL5, SIX3, LRP2, CTNNA2, MAP2, SRGAP3, RHOH, CDH10, FABP7, COL9A1, LRRN3, PTN, FEZF1, IRS4, CAPN6, HS6ST2, FZD3, RORB, CNTNAP3B, CNTNAP3, ACTN3, PAMR1, HPSE2, NELL2, LHX5, LM03, SLITRK1, SM0C1, CRABP1, RTN1, MT1F, CA4, CCBE1, NNAT, and MN1 preferably at least one selected from the group consisting of PAX6, HES5, and PAMR1 ; and/or
(iv) the gene specific for mesodermal cells may be at least one selected from the group consisting of HAND1, H0XB7, APLNR, LAPTM5, TNFRSF1B, GJA4, TIE1, ADGRL4, S1PR1, TBX15, PRRX1, ID3, EFNA1, GJ AS, FCN3, PLA2G2A, SHE, GBP2, GBP4, SAM Dll, DYNLT5, ADAMIS, GSTM3, CSRP1, TALI, DDR2, CYP1B1, EPAS1, CRIM1, CHMP3, HNMT, CNRIP1, GYPC, CALCRL, NAB1, COL3A1, DPP4, CYRIA, H0XD13, H0XD11, HOXDIO, H0XD9, COL6A3, CTDSP1, RASGRP3, FRMD4B, ARHGAP31, TM4SF1, TNFSF10, SST, PTH1R, WNT5A, PLSCR5, SEMA3G, IN KAI, MELTF, GATA2, MSX1, SLIT2, HOPX, LIMCH1, PARM1, MMRN1, FNIP2, VEGFC, UNC5C, HPGD, HAND2, IGFBP7, FAT4, GASK1B, KDR, TTC29, NFKB1, ISL1, HAPLN1, PDZD2, PITX1, TGFBI, AFAP1L1, HAND1, MSX2, ECSCR, SLIT3, CARTPT, MAST4, SLC26A2, F2RL2, DAB2, EBF1, TXNDC5, GF0D1, ADGRG6, BMP5, RSP03, TMEM200A, GSTA1, LST1, GPSM3, N0TCH4, ADGRF5, NT5E, PLAGL1, TWIST1, HGF, GNG11, DLX5, GIMAP4, CAV1, PCOLCE, H0XA7, H0XA9, H0XA11, H0XA13, H0XA3, THSD7A, HOXAIO, WNT2, STEAP1, SRPX, SYTL5, DIPK2B, BGN, NHS, ARMCX6, LPL, S0X7, SLC18A1, SCARA3, ADGRA2, BPNT2, HEY1, SNAI2, MATN2, RDH10, COLECI 0, RSP02, BAALC, TRPS1, ANGPT1, CTHRC1, LURAP1L, C0L15A1, PALM2AKAP2, NRARP, TEK, MYORG, IL33, NTRK2, CALCB, SLN, TENM4, ETS1, ESAM, OAF, FLI1, MS4A4A, SCN3B, ITGA8, ZNF503, UNC5B, HTRA1, PTPRE, IFIT3, BAMBI, MMRN2, VIM, CLEC1A, TMEM117, H0XC9, HOXCIO, H0XC11, TMCC3, KCNMB4, CPM, IGF1, LUM, EMP1, ACVRL1, PTPRB, CACNA1C, POSTN, LHFPL6, FLT1, PCDH9, TMEM255B, EFNB2, LPAR6, THSD1, D0CK9, RHOJ, CLEC14A, RNASE6, SLC39A2, DACT1, CRIP2, DLL4, ALDH1A2, ISLR, STRA6, L0XL1, MYZAP, IGDCC3, TM0D2, B2M, D0K4, F0XF1, CPNE2, CDH5, IL32, CRISPLD2, CDH13, IGFBP4, H0XB9, PRAC1, SP6, H0XB7, H0XB6, TBX4, TMEM100, H0XB13, PRCD, PECAM1, ICAM2, RAMP2, H0XB3, H0XB5, C0L1A1, SCARF1, RAB31, SERPINB8, LIPG, TSHZ1, APCDD1, PARD6G, CD93, TGM2, PROCR, PRND, RASSF2, SNAP25, PLVAP, EMP3, GMFG, DACT3, RRAS, RASIP1, KCTD15, TIMP3, CLDN5, JAM2, RCAN1, and ERG, preferably at least one selected from the group consisting of HANDl, H0XB7, and APLNR.
Surprisingly it was found that these genes are suitable for the determination in step e) of the method according to the present invention. They are specific for the respective differentiation (i.e., endodermal, ectodermal, mesodermal) or no differentiation (pluripotency), and provide a high sensitivity and specificity at the same time in each case. The genes are "specific for the respective differentiation (i.e., endodermal, ectodermal, mesodermal) or no differentiation (pluripotency)", because they are expressed at high levels only in the respective cells. Therefore, the results of the analysis of the respective gene transcripts may be used for the determination in step e) of the method according to the invention. According to the present invention, it is sufficient to amplify one gene specific for pluripotent cells (i.e., genes listed above under item (i)), one gene specific for endodermal cells (i.e., genes listed above under item (ii)), one gene specific for ectodermal cells (i.e., genes listed above under item (iii)), and one gene specific for mesodermal cells endodermal cells (i.e., genes listed above under item (iv)). It is also encompassed by the present invention that two or more genes for pluripotent cells are amplified, or that three or more genes for pluripotent cells are amplified, respectively. In a preferred embodiment, one or two genes specific for pluripotent cells are amplified. The same of course also applies to the genes for endodermal cells, ectodermal cells, and mesodermal cells. Again, for each of the cells one, two, three or more genes specific for the respective cell type can be amplified. Preferably, one or two genes are amplified for each of the cell types. Obviously, it is also encompassed by the present invention to amplify parts of the abovementioned genes.
If more than one gene or part thereof is amplified for one of the cell types, the amplification of each gene or part thereof can be performed subsequently or at the same time. It is preferably performed at the same time.
It was surprisingly found that especially CNMD, and/or NANOG, and/or SPP1 are suitable for the determination of pluripotency, i.e., the undifferentiated state, of the cells. They can be detected easily with high sensitivity. Accordingly, the analysis can be performed easily, and the result has a high accuracy.
In a preferred embodiment, the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1. In a further preferred embodiment, the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1. In a further preferred embodiment, the gene specific for mesodermal cells is selected from of HAND1, H0XB7, and/or APLNR. In a preferred embodiment of the method according to the present invention, the gene specific for pluripotent cells is selected from CNMD, NANOG, and/or SPP1, and/or the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1, and/or the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1, and/or the gene specific for mesodermal cells is selected from of HAND1, H0XB7, and/or APLNR. In still a further preferred embodiment of the method according to the present invention, the gene specific for pluripotent cells is selected from CNMD, NANOG, and/or SPP1, and the gene specific for endodermal cells is selected from GATA6, EOMES, and/or CER1, and the gene specific for ectodermal cells is selected from PAX6, HES5, and/or PAMR1, and the gene specific for mesodermal cells is selected from HANDl, H0XB7, and/or APLNR. It was shown that this combination of genes allows a determination of pluripotency and differentiation capacity of stem cells with high accuracy and high specificity.
The in vitro method of the present invention comprises amplification of genes or gene transcripts, respectively, specific for pluripotent cells, amplification of genes or gene transcripts, respectively, specific for endodermal cells, amplification of genes or gene transcripts, respectively, specific for ectodermal cells, and amplification of genes or gene transcripts, respectively, specific for mesodermal cells, or amplification of parts of the aforementioned genes or gene transcripts, respectively. Preferred primers are selected from:
(i) the first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 1 and 2, the pair SEQ ID NO: 3 and 4, and the pair SEQ ID NO: 5 and 6; and/or
(ii) the first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 7 and 8, the pair SEQ ID NO: 9 and 10, and the pair SEQ ID NO: 11 and 12; and/or (Hi) the first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 13 and 14, the pair SEQ ID NO: 15 and 16, and the pair SEQ ID NO: 17 and 18; and/or
(iv) the first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof may be a primer pair selected from the group consisting of the pair SEQ ID NO: 19 and 20, the pair SEQ ID NO: 21 and 22, and the pair SEQ ID NO: 23 and 24.
The preferred amplification primers according to SEQ ID NO: 1 and 2 enable for amplification of CNMD. The preferred amplification primers according to SEQ ID NO: 3 and 4 enable for amplification of NANOG. The preferred amplification primers according to SEQ ID NO: 5 and 6 enable for amplification of SPP1.
The preferred amplification primers according to SEQ ID NO: 7 and 8 enable for amplification of GATA6. The preferred amplification primers according to SEQ ID NO: 9 and 10 enable for amplification of EOMES. The preferred amplification primers according to SEQ ID NO: 11 and 12 enable for amplification of CER1.
The preferred amplification primers according to SEQ ID NO: 13 and 14 enable for amplification of PAX6. The preferred amplification primers according to SEQ ID NO: 15 and 16 enable for amplification of HES5. The preferred amplification primers according to SEQ ID NO: 17 and 18 enable for amplification of PAMR1.
The preferred amplification primers according to SEQ ID NO: 19 and 20 enable for amplification of HANDl. The preferred amplification primers according to SEQ ID NO: 21 and 22 enable for amplification of H0XB7. The preferred amplification primers according to SEQ ID NO: 23 and 24 enable for amplification of APLNR. In a preferred embodiment, the differentiation of iPSCs in step a) is effected via directed differentiation. Methods of performing directed differentiation are known to the person skilled in the art.
The samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells of step b) may be total RIMA isolated from the respective cells or molecules derived from total RNA isolated from the respective cells (e.g., cDNA). The samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells of step b) are preferably cDNA samples, preferably obtained by reverse transcription reaction from RNA isolated from the respective cells. Methods of isolating RNA from eukaryotic cells as well as methods of performing reverse transcription reactions of RNA are known to the person skilled in the art.
The amplification reactions performed in step d) of the method according to the present invention can be performed by any method known by the skilled artisan. They are preferably PCR methods such as quantitative polymerase chain reactions (qPCR) or digital polymerase chain reactions (dPCR).
Digital polymerase chain reaction (dPCR) is a biotechnological refinement of conventional polymerase chain reaction (PCR) methods that facilitates the precise quantification of nucleic acid concentrations. The key distinction between dPCR and traditional (semi)quantitative PCR, such as qPCR, lies in the approach to nucleic acid measurement. While qPCR provides only relative quantification, dPCR allows for real quantitative detection of nucleic acids. This is achieved by dividing reactions into thousands of individual compartments. This partitioning enables precise measurement, with each compartment yielding a binary result (positive or negative). In contrast to qPCR, which detects fluorescence intensity of intercalating dyes at each PCR cycle, dPCR is based on conventional end-point PCR coupled with fluorescence intensity measurement after the reaction is completed. The results obtained from step d) are further analysed in step e) of the method according to the present invention. The analysis allows to obtain a score, which ranges from 0 to 4. A score of 0 would indicate that the iPSCs have a bad integrity, e.g., are already differentiated and/or display impaired differentiation capacity. By taking each pluripotency/differentiation state (pluripotency, endoderm, ectoderm, mesoderm) into account, the integrity test is only successful if each individual subtest (pluripotency, endoderm, ectoderm, mesoderm) has been passed. The final score is obtained by adding the products of each individual pluripotency/differentiation score (quality parameter) with its certainty of classification p, yielding a maximum score of 4 for an unequivocal result (see Fig. 1 A). A score of 4 would thus indicate that all iPSCs are in a pluripotent state and can differentiate into each of the three primary germ layers. Preferably, the score is set such that a score between 3 and 4 in step e) indicates integrity of the iPSCs. As mentioned previously, this final score indicating the integrity of the iPSCs is also called "hiPSCore" in the following.
The object of the present application is further solved by providing a kit for determining the integrity of iPSCs, comprising a) a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof; b) a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof; c) a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof; d) a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof.
The amplification primers are preferably those described above for each of the different cell types. The object of the present application is further solved by an in vitro method for determining the status of differentiated iPSCs comprising the following steps: a) providing a differentiated iPSCs-derived sample; bl) mixing a first aliquot of the differentiated iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture; c) performing nucleotide amplification reaction of the amplification mixture and quantifying the amplification products; d) analysing the results of the quantification of the amplification products, thereby determining the status of the differentiated iPSCs.
The "status of differentiated iPSCs" refers to the question whether the iPSCs successfully differentiated into one of the primary germ layers endoderm, ectoderm, and mesoderm. By way of example, if iPSCs were differentiated to endodermal cells, a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof should be used in step bl), in order to determine whether the differentiated iPSCs are indeed endodermal cells.
The differentiated iPSCs-derived sample provided in step a) may be a sample prepared from differentiated iPSCs. Thus, the method may comprise as a first step the additional step 0) of preparing samples from differentiated iPSCs, thereby obtaining a differentiated iPSCs-derived sample. This differentiated iPSCs-derived sample of optional step 0) is the differentiated iPSCs-derived sample provided in step a).
The differentiated iPSCs-derived sample provided in step a) may be total RIMA isolated from the differentiated iPSCs or molecules derived from total RNA isolated from the differentiated iPSCs (e.g., cDNA). The differentiated iPSCs- derived sample provided in step a) is preferably a cDNA sample, preferably obtained by reverse transcription reaction from RIMA isolated from the differentiated iPSCs.
In step d) of the method, the results of the quantification of the amplification products are analysed and thereby, the status of the differentiated iPSCs is determined. By way of example, if the differentiated iPSCs-derived sample aliquot which was mixed with an amplification primer set capable of amplifying a gene specific for endodermal cells or a part thereof yields a positive result (indicating that the sample contains or is derived from endodermal cells), it can be concluded that the analysed sample of the differentiated iPSCs comprises endodermal cells. If the result is negative, it can be concluded that the analysed sample of the differentiated iPSCs does not comprise endodermal cells. The analysis allows to obtain a score, which ranges from 0 to 1. A score from 0 to 0.5 would indicate that the differentiated iPSCs do not have the expected/desired status. The score is obtained by multiplying the quality parameter obtained in the analysis with its certainty of classification p. A score from more than 0.5 to 1 would indicate that the differentiated iPSCs have achieved the expected/desired status.
This in vitro method for determining the status of differentiated iPSCs preferably comprises at least one of the following steps: b2) mixing a second aliquot of the differentiated iPSCs-derived sample with a second set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture; b3) mixing a third aliquot of the differentiated iPSCs-derived sample with a third set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, i.e., a part of said gene, thereby forming an amplification mixture. It is to be understood that "the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof" of the steps b2) and b3), respectively, is a gene specific for the same differentiation state as the differentiation state selected in step bl) of the in vitro method for determining the status of differentiated iPSCs.
The method may comprise one of the additional steps b2) and b3), or both of the additional steps b2) and b3). The order of the steps bl), b2), and b3) is not fixed. It is also possible that all steps bl), b2), and/or b3) are performed at the same time. The only requirement is that the steps bl), b2), and b3) are performed after step a) and before step c).
The problem underlying the present invention is further solved by an in vitro method for determining the status, especially the differentiation status, of iPSCs comprising the following steps: a) providing an iPSCs-derived sample; bla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2a) mixing a second aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3a) mixing a third aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4a) mixing a fourth aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; c) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; d) analysing the results of the quantification of the amplification products, thereby determining the status of the iPSCs. The method for determining the status of iPSCs of the present invention may be used to monitor the status of iPSCs obtained by reprogramming somatic cells during cultivation of said iPSCs in cell culture. Preferably, the status of the iPSCs may be the differentiation status of the iPSCs, which indicates whether a sample taken from an iPSCs cell culture is of the undifferentiated state (pluripotent), or of any of the three primary germ layers (endoderm, ectoderm, mesoderm), mixtures thereof, or none of the aforementioned states, e.g., is a further differentiated cell sample or a cell sample of non-iPSC origin. As described herein, iPSCs obtained by reprogramming somatic cells may, e.g., be used for patient-specific disease modelling or for treatment of patients with their own cells which have been treated ex vivo. However, during the "life cycle" of an iPSC, there may be periods in which the cells are only grown under controlled conditions (cell culture) and subcultured or passaged to prolong the lifespan and/or to increase the numbers of cells in culture whenever necessary. When iPSCs are cultured, new culture medium must be added at regular intervals, e.g., to maintain the pluripotency of the cells. If the culture medium is not changed at the appropriate time, the iPSCs may lose their pluripotency, i.e., they may lose their potential to differentiate into any of the three primary germ layers endoderm, ectoderm, and mesoderm. As a result, the iPSCs are not any more suitable to be used for patient-specific disease modelling or for treatment of patients with their own cells which have been treated ex vivo. Surprisingly, it has been found that it is possible to monitor the status of the above-described running cell cultures of iPSCs in a fast and easy way by the in vitro method for determining the status of iPSCs of the present invention. Thus, by analysing a sample taken from an iPSCs cell culture with the in vitro method for determining the status of iPSCs of the invention, it is possible to determine whether the iPSCs of the said cell culture are still pluripotent and may be used for patient-specific disease modelling, etc.
The iPSCs-derived sample provided in step a) may be prepared from cells taken from an iPSCs cell culture grown under controlled conditions, e.g., an ordinary or normal iPSCs cell culture. For example, the cells used to prepare the iPSCs- derived sample may be taken from said cell culture directly before or after passaging, or after a longer cultivation interval in which the cells did not receive new culture medium (e.g., a weekend). Thus, the method may comprise as a first step the additional step 0) of preparing samples from an iPSCs cell culture, thereby obtaining an iPSCs-derived sample. This iPSCs-derived sample of optional step 0) is the iPSCs-derived sample provided in step a). The iPSCs- derived sample provided in step a) may also be derived from other cells, provided that the said cells originate from iPSCs.
The iPSCs-derived sample provided in step a) may be total RIMA isolated from the cells taken from the iPSCs cell culture or molecules derived from total RNA isolated from cells taken from the iPSCs cell culture (e.g., cDNA). The iPSCs- derived sample provided in step a) is preferably a cDNA sample, preferably obtained by reverse transcription reaction from RNA isolated from the cells taken from the iPSCs cell culture.
The order of the steps bla), b2a), b3a), and b4a) in the method of the invention is not fixed. It is possible that step b2a) is performed before step bla), that step b3a) is performed before step bla), and so on. It is also possible that all steps bla), b2a), b3a), and b4a) are performed at the same time. The only requirement is that the steps bla), b2a), b3a), and b4a) are performed after step a) and before step c) of the method of the invention. It is also possible that only one of the steps bla), b2a), b3a), and b4a) is performed in the method of the invention after step a) and before step c).
In step d) of the method, the results of the quantification of the amplification products are analysed and thereby, the status of the iPSCs is determined. By way of example, if the iPSCs-derived sample aliquot which was mixed with the amplification primer set capable of amplifying a gene specific for pluripotent cells or a part thereof yields a positive result (indicating that the sample contains or is derived from pluripotent cells), it can be concluded that the analysed sample of the iPSCs comprises pluripotent cells. If the result is negative, it can be concluded that the analysed sample of the iPSCs does not comprise pluripotent cells. The analysis allows to obtain a score, which in the case that only one of the steps bla), b2a), b3a), and b4a) was performed ranges from 0 to 1. A score from 0 to 0.5 would then indicate that the cells do not have the expected/desired status. A score from more than 0.5 to 1 would indicate that the cells have achieved the expected/desired status. When all the steps bla), b2a), b3a), and b4a) are performed, the total score would be from 0 to 4, because each of the four subtests (pluripotent, endoderm, ectoderm, mesoderm) would lead to a score between 0 and 1, and the respective subtest scores are summed up. The respective subtest scores are obtained by multiplying the quality parameter obtained in each subtest with its certainty of classification p.
This in vitro method for determining the status of iPSCs preferably additionally comprises at least one of the following steps: bib) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture. The method of the invention may comprise one of the additional steps bib), b2b), b3b), and b4b), two of the additional steps bib), b2b), b3b), and b4b), three of the additional steps bib), b2b), b3b), and b4b), or four of the additional steps bib), b2b), b3b), and b4b).
The order of the additional steps bib), b2b), b3b), and b4b) in the method of the invention is not fixed. It is, e.g., possible that additional step bib) is performed after step bla) or before step bla). It is also possible that step bib) is performed after step b4a), and so on. It is also possible that all steps bla), b2a), b3a), b4a), bib), b2b), b3b), and b4b) are performed at the same time. The only requirement is that the additional steps bib), b2b), b3b), and b4b) - like the steps bla), b2a), b3a), and b4a) - are performed after step a) and before step c) of the method of the invention.
This in vitro method for determining the status of iPSCs preferably additionally comprises at least one of the following steps: blc) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
The method of the invention may comprise one of the additional steps blc), b2c), b3c), and b4c), two of the additional steps blc), b2c), b3c), and b4c), three of the additional steps blc), b2c), b3c), and b4c), or four of the additional steps blc), b2c), b3c), and b4c).
The order of the additional steps blc), b2c), b3c), and b4c) in the method of the invention is not fixed. It is, e.g., possible that additional step blc) is performed after step bla), before step bla), after step bib), or before step bib). It is also possible that step blc) is performed after step b4a) or after step b4b), and so on. It is also possible that all steps bla), b2a), b3a), b4a), bib), b2b), b3b), b4b), blc), b2c), b3c), and b4c) are performed at the same time. The only requirement is that the additional steps blc), b2c), b3c), and b4c) - like the steps bla), b2a), b3a), b4a), bib), b2b), b3b), and b4b) - are performed after step a) and before step c) of the method of the invention.
The problem underlying the present invention is further solved by a computer- implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample, the method comprising the following steps: providing an iPSC-derived sample to be analysed; performing a PCR analysis on the iPSC-derived sample and obtaining fluorescence signal-derived values for n cycles of the PCR.; determining a quality parameter for the iPSC-derived sample using a trained classifier and the obtained fluorescence signal- derived value.
In the computer-implemented method of the invention, the fluorescence signal- derived value is used as an input for the classifier. The fluorescence signal- derived value may comprise an intensity value for each cycle of the PCR.. The classifier may be based on a Support Vector Machine (SVM), Random Forest or an Artificial Neural Network (ANN). The quality parameter may be a binary parameter having values of 0 (e.g., indicating that the sample does not contain or is not derived from pluripotent cells, or indicating that the sample does not contain or is not derived from endodermal cells) and 1 (e.g., indicating that the sample contains or is derived from pluripotent cells, or indicating that the sample contains or is derived from endodermal cells). The trained classifier may be trained with training data comprising pairs of fluorescence signal-derived values and classification data corresponding to each fluorescence signal-derived value and characterising the integrity of an iPSC-derived sample.
The quality of the iPSC-derived sample may allow a statement about the pluripotency status of the iPSCs and/or the differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm. Quality parameters of each of the differentiation states can be used to subsequently determine a score indicating the integrity of the iPSCs. This score is called "hiPSCore". To calculate the hiPSCore, the quality parameter of each of the differentiation states (subtests) is multiplied with its certainty of classification p, and the resulting products are summed up, yielding the hiPSCore (see Fig. 1 A). It is to be understood that, regardless of the number of genes used for classification, only a single quality parameter and a single certainty of classification for a given subtest are obtained. In a similar way, the quality parameters can be used to subsequently determine the status of differentiated iPSCs (in the in vitro method for determining the status of differentiated iPSCs of the present invention), or to subsequently determine the status, especially the differentiation status, of iPSCs (in the in vitro method for determining the status, especially the differentiation status, of iPSCs of the present invention).
The iPSCs-derived sample to be analysed may be total RIMA isolated from pluripotent cells or from iPSCs differentiated into endodermal cells, into ectodermal cells, or into mesodermal cells, or may be molecules derived from total RNA isolated from pluripotent cells or from iPSCs differentiated into endodermal cells, into ectodermal cells, or into mesodermal cells (e.g., cDNA). The iPSC-derived sample to be analysed may be cDNA samples, preferably cDNA samples from pluripotent cells or from iPSCs differentiated into endodermal cells, into ectodermal cells, or into mesodermal cells. The cDNA samples are preferably obtained by reverse transcription reaction from isolated total RIMA of the respective cells.
The PCR analysis performed on the iPSC-derived sample may be a quantitative polymerase chain reaction (qPCR) analysis or a digital polymerase chain reaction (dPCR) analysis. Preferably, a qPCR analysis is performed, and the fluorescence signal-derived values obtained for n cycles of the PCR are the cycle threshold (Ct) values, i.e., the cycle at which the signal intensity significantly crosses the background signal, obtained by the qPCR device.
Preferably, the computer-implemented method of the present invention is characterised by the following: providing a data set comprising fluorescence signal-derived values and classification data, wherein the classification data assigns a quality parameter to each of the fluorescence signal- derived values; splitting the data set into a first group of data comprising N pairs of fluorescence signal-derived values and corresponding quality parameters, wherein the first group comprises pl percent of the entire data set, and a second group of data comprising M pairs of fluorescence signal-derived values and corresponding quality parameters, wherein the second group comprises p2 percent of the entire data set, the sum of pl and p2 being 100; using the first group of data for training c classifiers and using the second group of data for testing the c classifiers, obtaining a rating parameter n for each of the c classifiers, wherein each rating parameter n indicates the performance of each of the c classifiers; and selecting from the c classifiers the classifier with the highest rating for analysing the iPSC-derived sample to be analysed. The rating may define the accuracy of the classifier or e.g., the speed of the classification process. Thus, different classifiers can be tested, and the best classifier can be used for the classification of the fluorescence signal-derived value. The different classifiers tested may be e.g., an ANN, a CNN and a SVM. Alternatively, the different classifiers can be of the same type (such as an SVM), while the parameters of the classifier are varied in order to obtain the optimum classifier, pl may be 70 and p2 may be 30.
An exemplary embodiment of the computer-implemented method of the present invention is shown in Fig. 2 A. Fig. 2 B shows an example for the fluorescence signal-derived values which may be used in the computer-implemented method of the invention.
The problem underlying the present invention is further solved by a computer- readable storage medium comprising instructions which, when executed by a computer, cause the computer to carry out the computer-implemented method of the present invention.
It is to be understood that preferred embodiments or preferred features mentioned for one embodiment are also to be applied in the same way for all other embodiments mentioned in the application. Each preferred embodiment and each preferred feature can be combined with each other without further limitation.
In the following example, the present invention is disclosed but without limiting the scope of protection to this specific example. Example
To determine the integrity of iPSCs in an iPSCs sample with unknown pluripotency status and differentiation capacity, a score that allows to assess the integrity of the iPSCs was determined. The said score is called "hiPSCore" and may enable standardisation of iPSC integrity testing.
To streamline the analysis and increase multiplexing capacity, a quality parameter was determined based on the assessment of each differentiation state (i.e., pluripotency, endoderm, ectoderm, mesoderm) with its corresponding marker gene set individually. The quality parameter for each of the differentiation states was used to calculate the said "hiPSCore".
Details of the culturing of cells, especially iPSCs, and how their integrity is determined as well as calculation of the hiPSCore are described in more detail hereinafter.
Cell culture
Human iPSCs, purchased from Cell Applications, were grown in mTeSR. plus complete medium on 6-well plates coated with growth factor-reduced Geltrex.
Directed differentiation
Directed differentiation of human iPSCs into the three primary germ layers was performed by employing two different commercially available kits, namely the StemMACS Trilineage Differentiation kit (Miltenyi Biotec) and the StemDIFF Trilineage Differentiation kit (STEMCELL Technologies) according to the manufacturers' instructions. Accordingly, 5 * 10^4 (mesoderm)/2 * 10^5 (endoderm, ectoderm) iPSCs were seeded for differentiation in Geltrex-coated wells of a 24-well plate. After five (STEMCELL: endoderm, mesoderm) and seven days (rest) of differentiation, the cells were washed twice with PBS and harvested into 300 pl Trizol (Thermo). RNA isolation, cDNA transcription and qPCR
Total RIMA isolation was performed using the Direct-zol RNA Miniprep kit (Zymo) including on-column DNAse digestion according to the manufacturers' instructions. RNA was eluted in 25 pl aqua dest. and concentration measured on a NanoDrop2000 spectrophotometer (Thermo). 1 pg of RNA was reverse transcribed using the HiScript III RT SuperMix for qPCR kit (Vazyme). Afterwards, cDNA was diluted 1/10 with aqua dest. and used in a 10 pl qPCR reaction consisting of 5 pl ChamQ Universal SYBR qPCR Master Mix (Vazyme), 3 pl aqua dest., 1 pl primer Mix (forward + reverse @ 2.5 pM each for a final primer concentration of 250 nM), and 1 pl diluted cDNA sample. Reactions were run on QuantStudio 3 (Thermo) with a fast qPCR programme (3 sec denaturation, 20 sec annealing and extension) for 40 cycles followed by a Melt Curve analysis. Gene expression was normalized to the mean of two reference genes (ACt).
Double-stranded cDNA preparation and amplification
For nanopore long read sequencing, 200 ng of total RNA of two biological replicates of iPSC pluripotent control cells and cells differentiated with the StemMACS Trilineage differentiation kit (Miltenyi) were used for first strand cDNA synthesis in a 6 pl reaction consisting of 2 pl 10 pM RT primer (5'->3': AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TV; SEQ ID NO: 25; The "V" in the primer sequence stands for "not T", so can be any DNA base other than T (A, C, G). This primer is a degenerated primer to prevent poly(A) slippage. Poly(A) slippage is caused by the mRNA poly(A) tail which can cause mistakes during reverse transcription. This is prevented by degenerate primers adding a "non-T" to the primer binding to poly(A).), 1 pl dNTPs (10 mM each, Vazyme) and aqua dest. for 5 min at 70 °C.
Second strand synthesis was performed by adding 2.5 pl Template Switching Buffer (New England Biolabs), 0.5 pl 75 pM Template Switching Oligo (5'->3': GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG; SEQ ID NO: 26; The "rG" in the primer sequence stand for the RNA base guanine.) and 1 pl Template Switching RT Enzyme Mix (New England Biolabs) to the first strand reaction and incubating 90 min at 42 °C, followed by heat inactivation at 85 °C for five minutes. Afterwards, 10 pl double-stranded cDNA were subjected to cDNA amplification by adding 25 pl Phanta Max Buffer (Vazyme), lpl dNTPs (10 mM each, Vazyme), 2pl Phanta Max Super Fidelity Polymerase (Vazyme), 1 pl 10 pM cDNA PCR Primer (5' -> 3': AAG CAG TGG TAT CAA CGC AGA GT; SEQ ID NO : 27), and 11 pl aqua dest. and performing a 7 cycles PCR reaction. Afterwards, 1 pl Exonuclease I (Thermo) was directly added to the PCR reaction, and incubated for 15 min at 37 °C, followed by heat inactivation for 15 min at 80 °C. Final products were purified using AMPure XP (Beckman Coulter) beads at a ratio of 0.8 X PCR product to beads according to the manufacturers' instructions. Double-stranded cDNA concentration was measured on a Qubit Fluorometer (Thermo) using the dsDNA high sensitivity (HS) kit (Thermo).
Nanopore long read sequencing
Library preparation was performed according to the Ligation sequencing amplicons - Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies, SQK- NBD114.24) according to the version NBA_9168_vl l4_revE_15Sep2022. Accordingly, equal amounts of double-stranded cDNA (20.25 ng) were subjected to library preparation. The final library was quantified on a Qubit Fluorometer (Thermo) using the dsDNA HS kit (Thermo). Library size was determined using a D1000 Screen Tape assay (Agilent). A final amount of 15 fmol was loaded on a P2 solo flow cell and sequenced for 72 h.
Sequencing data analysis
Basecalling was performed using the Guppy basecaller (v.6.4.6) with a minimum quality of 5. FASTQ files were concatenated and mapped to the reference genome (GRCh38) using Minimap2 (v.2.24) with the options (-uf -kl4) used for noisy direct RNA seq ONT reads to enhance sequence retrieval. All further analysis was performed using R (v.4.2.2). Sequence reads were counted using Rsubread featurecounts with options for long sequencing reads and GRCh38.106 to annotate transcripts. Afterwards, reads were normalized to gene counts per million using the R package edgeR (v.3.40.2), Bioconductor. Differentially expressed transcripts were assessed by normalizing Iog2 counts to the pluripotent samples and filtering for high Iog2 fold changes (I2fc, depending on the differentiation state higher than 3 to 6 for differentiated samples and lower than -2 for each differentiated sample vs the pluripotent controls). All data was visualized using ggplot2 (v.3.4.2) and ComplexHeatmap (v.2.14).
Assessment of the integrity of iPSCs
In preparation of determination of each of the quality parameters, different machine learning-based models were tested to identify the model with the highest predictive accuracy and specificity for each differentiation state. The process of the machine learning and comparison of the models as well as determination of the quality parameter of each differentiation sate is described in more detail hereinbelow.
To train the machine learning algorithm, data on 15 different induced pluripotent stem cell (iPSC) lines was obtained. To that end, gene expression for the 12 target genes (three per differentiation state; pluripotent: CNMD, NANOG, SPP1; endoderm: GATA6, EOMES, CER1; ectoderm: PAX6, HES5, PAMR1; mesoderm: HAND1, H0XB7, APLNR) was analysed in the basal pluripotent state, and in the cells differentiated into each of the three primary germ layers, endoderm, ectoderm, and mesoderm via qPCR. This data was normalised to the mean Cycle threshold (Ct) of two reference genes GAPDH and ACTS'). The normalised data from the qPCR experiments of pluripotent and differentiated iPSCs for each of the 12 target genes was taken as input for training the classification model. Sample quality was annotated by a binary dummy-coded classification system as an input as "1" (positive/good quality) or "0" (negative/bad quality) according to the following rules:
Pluripotency: "1" for known pluripotent samples, and "0" for known differentiated samples
Endoderm : "1" for known endoderm samples, and "0" for known pluripotent or ectoderm or mesoderm samples
Ectoderm : "1" for known ectoderm samples, and "0" for known pluripotent or endoderm or mesoderm samples
Mesoderm: "1" for known mesoderm samples, and "0" for known pluripotent or endoderm or ectoderm samples.
To increase data availability for machine learning, each cell line was differentiated into the three primary germ layers by two different commercially available trilineage differentiation protocols. This resulted in a total of 1,296 datapoints. The R package caret (Kuhn, 2008) was used as a framework to train, tune, and select the optimal models for each germ layer. A variety of models, including different random forest and neural network implementations, were tested. 70% of the obtained data were used as the input to predict the remaining 30% of the data. For model validation, repeated cross validation was used (k- fold splits: 10; repeats: 3). To account for class imbalances due to unequal distribution of quality scores ("1" vs "0"), different sampling techniques (downscaling, upscaling, ROSE, SMOTE) were applied and selected based on the optimal model performance determined by ROC-AUC (receiver operating characteristic curve - area under curve). The tuning parameters for the final models are given in Table 1 and the final selected models in Table 2. Table 1 : Sampling and Model tuning parameters of the tested and optimised models.
Figure imgf000043_0001
Table 2: Final selected models based on the ROC-AUC performance.
Figure imgf000044_0001
The so called hiPSCore web application, a classification algorithm using the machine learning-based model with the highest predictive accuracy and specificity for each differentiation state (see Table 2), was coded using the R Shiny (v.1.7.4) framework and custom functions were implemented to predict the hiPSCore, i.e., the score that allows to assess the integrity of the iPSCs, of individual samples based on the input according to the nucleotide amplification reactions of the individual samples.
The final hiPSCore is calculated by taking the quality parameter of either "0" or "1" of each germ layer, which is determined with the respective machine learning-based model with the highest predictive accuracy and specificity for the respective germ layer, and multiplying it with the certainty of classification p. It is to be understood that, regardless of the number of genes used for classification, only a single quality parameter and a single certainty of classification for a given subtest are obtained. Subsequently the resulting values are added up, yielding a continuous quality score with: > 3: pass, > 2: warning, < 2: fail (see Fig. 1 A). This continuous quality score ("hiPSCore") can be reported enabling easy sharing of quality assessment across laboratories and cell lines. It is important to note that a sample will only pass the test if it shows positive pluripotency gene expression patterns and positive gene expression patterns for each of the differentiated germ layers. This ensures that only truly pluripotent samples will be classified. The whole method for determining the integrity of iPSCs of the present invention will be explained with reference to Figs. 1 B and C. As shown in Fig. 1 B, in a first step, an iPSCs sample is provided. Samples of this iPSCs sample are differentiated into the three primary germ layers (endoderm, ectoderm, mesoderm). One aliquot of the iPSCs is not differentiated, yielding in total four samples (undifferentiated iPSCs, endodermal cells, ectodermal cells, and mesodermal cells). In a next step (not shown), the total RIMA of each of the four cell samples is isolated and reversely transcribed into cDNA. Thus, samples are prepared from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells. In a next step (not shown), aliquots of the iPSCs-derived cDNA, the endodermal cells-derived cDNA, the ectodermal cells-derived cDNA, and the mesodermal cells-derived cDNA are mixed with a set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, a set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, a set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, and a set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, respectively. The resulting amplification mixtures are subjected to qPCR nucleotide amplification reactions and the resulting respective amplification products are quantified ("hiPSCore qPCR Assay" shown in Fig. 1 B). In the last step, the results of the quantification of the amplification products are analysed with the "hiPSCore web application" ("classification" shown in Fig. 1 B), thereby obtaining the score that allows to assess the integrity of the iPSCs, i.e., the hiPSCore (for reference see also Fig. 1 A). As can be seen in Fig. 1 B, in the case of Sample 1, all the four cell types pluripotent (undifferentiated), endoderm, ectoderm, and mesoderm were present and thus, all four samples gave a positive result. The hiPSCore is above 3 and the integrity of the iPSCs is confirmed. However, in the case of Sample 2, only endodermal, ectodermal, and pluripotent (undifferentiated) cells were present and gave a positive result. The result for mesodermal cells was negative. The hiPSCore is below 3 and integrity of the iPSCs is not confirmed. Another example for different hiPSCores is shown in Fig. 1 C. On the left panel, it can be seen that only the iPSCs-derived sample gave a positive result (amplification product of genes specific for pluripotent cells was present at high levels in all three analysed samples for pluripotent cells), while the endodermal cells-derived sample, the ectodermal cells-derived sample, and the mesodermal cells-derived sample gave a negative result (no amplification product of genes specific for mesodermal cells, of genes specific for endodermal cells, and of genes specific for ectodermal cells was present at high levels in all analysed samples). The hiPSCore was 0.94, indicating that the integrity of the iPSCs is not guaranteed. On the middle panel, it can be seen that the mesodermal cells- derived sample gave a negative result (no amplification product of genes specific for mesodermal cells was present), while the endodermal cells-derived sample, the ectodermal cells-derived sample, and the iPSCs-derived sample gave a positive result (amplification product of genes specific for the respective cell types was present at high levels). The hiPSCore was 2.98, indicating that the integrity of the iPSCs may be compromised. On the right panel, it can be seen that the iPSCs-derived sample, the endodermal cells-derived sample, the ectodermal cells-derived sample, and the mesodermal cells-derived sample gave a positive result (amplification product of genes specific for the respective cell types was present at high levels). The hiPSCore was 3.61, indicating that the integrity of the iPSCs is guaranteed.
A correlation matrix, as depicted in Fig. 3, illustrates the relationships between the amplified genes and the state of the respective cell status (pluripotency, endoderm, ectoderm, mesoderm). A correlation value of 1 represents a perfect positive correlation indicating a very high similarity between two samples. On the other hand, a value of -1 represents a perfect negative correlation indicating no similarity between two samples. A value of 0 represents no correlation indicating no relationship between two samples at all. Sequence listing
SEQ ID NO: 1
CCGTGACCAAACAGAGCATCTC
SEQ ID NO: 2
CTGTTGTCCTTCACAGGCTGATC
SEQ ID NO: 3
TCCAACATCCTGAACCTCAG
SEQ ID NO: 4
ACCATTGCTATTCTTCGGCC
SEQ ID NO: 5
CGAGGTGATAGTGTGGTTTATGG
SEQ ID NO: 6
GCACCATTCAACTCCTCGCTTTC
SEQ ID NO: 7
TGTGCGTTCATGGAGAAGATCA
SEQ ID NO: 8
TTTGATAAGAGACCTCATGAACCGACT
SEQ ID NO: 9
GGCTGTCTCCTAGCAACTCC
SEQ ID NO: 10
GCATAATACCCTCCCATGCCT SEQ ID NO: 11
CCCATCAAAAGCCATGAAGT
SEQ ID NO: 12
AATGAACAGACCCGCATTTC
SEQ ID NO: 13
GAGGTCAGGCTTCGCTAATG
SEQ ID NO: 14
TTGCTTGAAGACCACAATGG
SEQ ID NO: 15
CTGCTCAGCCCCAAAGAG
SEQ ID NO: 16
GCTCGATGCTGCTGTTGAT
SEQ ID NO: 17
TTGCCAGCAGAATGGAGAGTGG
SEQ ID NO: 18
CTTGACTGAACCTGCATCGGAAG
SEQ ID NO: 19
ACATCGCCTACCTGATGGAC
SEQ ID NO: 20
CGGCTCACTGGTTTAACTCC SEQ ID NO: 21
ATCTACCCCTGGATGCGAAGCT
SEQ ID NO: 22
GCGTCAGGTAGCGATTGTAGTG
SEQ ID NO: 23
TTGCAGAGTGGGTGACAGAG
SEQ ID NO: 24
CTGGTTGTCTGCCCCATAGT
SEQ ID NO: 25
AAGCAGTGGTATCAACGCAGAGTAC I I I I I I I I I I I I I I I I I I I I I I I I I I I I I TV
SEQ ID NO: 26
GCTAATCATTGCAAGCAGTGGTATCAACGCAGAGTACATrGrGrG
SEQ ID NO: 27
AAGCAGTGGTATCAACGCAGAGT

Claims

Claims
1. In vitro method for determining the integrity of induced pluripotent stem cells (iPSCs) comprising the following steps: a) differentiating samples of the iPSCs into endodermal, ectodermal, and mesodermal cells; b) preparing samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells; cla) mixing a first aliquot of an iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2a) mixing a first aliquot of an endodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3a) mixing a first aliquot of an ectodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4a) mixing a first aliquot of a mesodermal cells-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; d) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; e) analysing results of the quantification in step d) of the amplification products, thereby obtaining a score that allows to assess the integrity of the iPSCs.
2. The method according to claim 1, wherein the method additionally comprises at least one of the following steps: clb) mixing a second aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2b) mixing a second aliquot of the endodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3b) mixing a second aliquot of the ectodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4b) mixing a second aliquot of the mesodermal cells-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
3. The method according to claim 2, wherein the method additionally comprises at least one of the following steps: clc) mixing a third aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; c2c) mixing a third aliquot of the endodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; c3c) mixing a third aliquot of the ectodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; c4c) mixing a third aliquot of the mesodermal cells-derived sample with a third set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
4. The method according to any one of claims 1 to 3, wherein the integrity of the iPSCs is a pluripotency status of the iPSCs and/or a differentiation capacity of the iPSCs into the primary germ layers endoderm, ectoderm, and mesoderm.
5. The method according to any one of claims 1 to 4, wherein the iPSCs are human induced pluripotent stem cells (hiPSCs).
6. The method according to any one of claims 1 to 5, wherein
(i) the gene specific for pluripotent cells is at least one selected from the group consisting of CNMD, NANOG, SPP1, DHRS3, L1TD1, TMEM125, SYT14, SMPDL3B, LCK, SF3B4, PDPN, RAB42, VASH2, RABGAP1L, VENT5B, VSNL1, ADD2, AP1S3, B3GNT7, UGP2, CRYGD, TUBA4A, KHK, CHAC2, CDCP1, GPD1L, TIMP4, IFRD2, CAMKV, DPPA4, SYNPO2, ZFP42, RASL11B, APELA, TRIML2, NSG1, CXCL5, KLKB1, SCGB3A2, POLR3G, NOP16, HRH2, PAK1IP1, PSMB8, PSMB9, POU5F1, TBRG4, MACC1, MAGI2, RARRES2, CCL26, DIAPH2, PIM2, ZMAT4, PRDM14, FABP5, RRS1, BOP1, TERFI, IDO1, ENSG00000258417, HHLA1, COL14A1, POU5F1B, E2F8, ZNF483, ZNF215, GRIA4, FAM111B, FOSL1, KCTD14, GLB1L3, FGF19, NRIP3, CUZD1, CCDC172, HTR7, ZNF485, TEAD4, SLC38A4, PHC1, METTL1, SLC6A15, MGAT4C, HSPH1, EDNRB, CCDC169, DCLK1, MTHFD1, EGLN3, GPR176, RBPMS2, AEN, CKMT1B, CKMT1A, MT1X, NANOGP8, SCG3, GDPGP1, VAT1L, DDX28, CHST4, ALG1, SYNGR3, C1QBP, PIGW, PPP1R1B, PIPOX, RBFOX3, HMSD, FERMT1, TNNC2, DNMT3B, FXYD7, CYP2S1, TOMM40, POLR1G, PPM IN, SYN3, and MCM5, preferably at least one selected from the group consisting of CNMD, NANOG, and SPP1 ; and/or
(ii) the gene specific for endodermal cells is at least one selected from the group consisting of GATA6, EOMES, CER1, F3, PIFO, AGL, PDZK1, CD48, DUSP10, ITLN2, KIAA0040, USH2A, FYB2, KCNK1, RAB25, APOA2, SLC5A9, MIXL1, LEFTY1, LEFTY2, FMN2, S100A16, S100A14, ELF3, CAPN13, LRP1B, KCNJ3, STAT4, PELI1, KCNK12, TTN, CCDC141, RABI 7, CLIP4, VI LI, VAMP8, COBLL1, CD80, UPK1B, TAGLN3, CLDN18, EHHADH, CLDN11, PLSCR1, RARB, TRMT44, ETN PPL, TMPRSS11E, PPM IK, AADAT, ACOX3, MFAP3L, CPE, ACSL1, ANXA3, GPM6A, RASSF6, PLCXD3, FST, CLVS2, TRDN, GMPR, SAMD3, GMPR, EGFL8, MANEA, HLA-DQB1, VEGFA, SEMA3E, CALCR, KIAA0895, EPHB6, KEL, STEAP1B, CASTOR2, PRSS1, PRSS2, PLXNA4, GRPR, NR0B1, KLF8, CLTRN, PORCN, ARSL, STC1, KIF13B, TRPA1, LY6E, FAM135B, CSMD3, FGF17, PIP5K1B, CTSV, TTC39B, GALNT12, PTGDS, DOCK8, CCKBR, DSCAML1, CST6, OR52A1, OVCH2, GRAM DIB, MYO3A, CPY26A1, PLAC9, IGFBP6, TESPA1, CNNM1, SLC2A3, NTN4, NUDT4, LGR5, SLC2A14, LMNTD1, ACSM4, DNAJC15, KLF5, F10, EPSTI1, GSC, 0TX2, UNC13C, PIEZ01, YPEL3, PMFBP1, HP, ARL4D, NTN1, MFAP4, TVP23C, PIK3R5, KRT19, CELF4, RNF152, POTEC, F0XA2, WFD2C, CST1, DBNDD2, HNF4A, TLE2, NCR1, NLRP7, CD70, TNFSF14, HPN, APOE, SULT2B1, GIPR, APOCI, C3, RIPPLY3, DSCAM, and ERVH48-1, preferably at least one selected from the group consisting of GATA6, EOMES, and CER1 ,- and/or
(iii) the gene specific for ectodermal cells is at least one selected from the group consisting of PAX6, HESS, PAMR1, DLK1, P0U3F1, ATP1A2, SYT11, DPYSL5, SIX3, LRP2, CTNNA2, MAP2, SRGAP3, RHOH, CDH10, FABP7, COL9A1, LRRN3, PTN, FEZF1, IRS4, CAPN6, HS6ST2, FZD3, RORB, CNTNAP3B, CNTNAP3, ACTN3, PAMR1, HPSE2, NELL2, LHX5, LM03, SLITRK1, SM0C1, CRABP1, RTN1, MT1F, CA4, CCBE1, NNAT, and MN1 preferably at least one selected from the group consisting of PAX6, HES5, and PAMR1 ; and/or
(iv) the gene specific for mesodermal cells is at least one selected from the group consisting of HAND1, H0XB7, APLNR, LAPTM5, TNFRSF1B, GJA4, TIE1, ADGRL4, S1PR1, TBX15, PRRX1, ID3, EFNA1, GJ AS, FCN3, PLA2G2A, SHE, GBP2, GBP4, SAM Dll, DYNLT5, ADAMIS, GSTM3, CSRP1, TALI, DDR2, CYP1B1, EPAS1, CRIM1, CHMP3, HNMT, CNRIP1, GYPC, CALCRL, NAB1, COL3A1, DPP4, CYRIA, H0XD13, H0XD11, HOXDIO, H0XD9, COL6A3, CTDSP1, RASGRP3, FRMD4B, ARHGAP31, TM4SF1, TNFSF10, SST, PTH1R, WNT5A, PLSCR5, SEMA3G, IN KAI, MELTF, GATA2, MSX1, SLIT2, HOPX, LIMCH1, PARM1, MMRN1, FNIP2, VEGFC, UNC5C, HPGD, HAND2, IGFBP7, FAT4, GASK1B, KDR, TTC29, NFKB1, ISL1, HAPLN1, PDZD2, PITX1, TGFBI, AFAP1L1, HAND1, MSX2, ECSCR, SLIT3, CARTPT, MAST4, SLC26A2, F2RL2, DAB2, EBF1, TXNDC5, GF0D1, ADGRG6, BMP5, RSPO3, TMEM200A, GSTA1, LST1, GPSM3, N0TCH4, ADGRF5, NT5E, PLAGL1, TWIST1, HGF, GNG11, DLX5, GIMAP4, CAV1, PCOLCE, H0XA7, H0XA9, H0XA11, H0XA13, H0XA3, THSD7A, HOXAIO, WNT2, STEAP1, SRPX, SYTL5, DIPK2B, BGN, NHS, ARMCX6, LPL, S0X7, SLC18A1, SCARA3, ADGRA2, BPNT2, HEY1, SNAI2, MATN2, RDH10, COLECI 0, RSPO2, BAALC, TRPS1, ANGPT1, CTHRC1, LURAP1L, COL15A1, PALM2AKAP2, NRARP, TEK, MYORG, IL33, NTRK2, CALCB, SLN, TENM4, ETS1, ESAM, OAF, FLU, MS4A4A, SCN3B, ITGA8, ZNF503, UNC5B, HTRA1, PTPRE, IFIT3, BAMBI, MMRN2, VIM, CLEC1A, TMEM117, H0XC9, HOXCIO, H0XC11, TMCC3, KCNMB4, CPM, IGF1, LUM, EMP1, ACVRL1, PTPRB, CACNA1C, POSTN, LHFPL6, FLT1, PCDH9, TMEM255B, EFNB2, LPAR6, THSD1, D0CK9, RHOJ, CLEC14A, RNASE6, SLC39A2, DACT1, CRIP2, DLL4, ALDH1A2, ISLR, STRA6, L0XL1, MYZAP, IGDCC3, TM0D2, B2M, D0K4, F0XF1, CPNE2, CDH5, IL32, CRISPLD2, CDH13, IGFBP4, H0XB9, PRAC1, SP6, H0XB7, H0XB6, TBX4, TMEM100, H0XB13, PRCD, PECAM1, ICAM2, RAMP2, H0XB3, H0XB5, C0L1A1, SCARF1, RAB31, SERPINB8, LIPG, TSHZ1, APCDD1, PARD6G, CD93, TGM2, PROCR, PRND, RASSF2, SNAP25, PLVAP, EMP3, GMFG, DACT3, RRAS, RASIP1, KCTD15, TIMP3, CLDN5, JAM2, RCAN1, and ERG, preferably at least one selected from the group consisting of HANDl, H0XB7, and APLNR.
7. The method according to any one of claims 1 to 6, wherein
(i) the first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof is a primer pair selected from the group consisting of the pair SEQ ID NO: 1 and 2, the pair SEQ ID NO: 3 and 4, and the pair SEQ ID NO: 5 and 6; and/or
(ii) the first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof is a primer pair selected from the group consisting of the pair SEQ ID NO: 7 and 8, the pair SEQ ID NO: 9 and 10, and the pair SEQ ID NO: 11 and 12; and/or
(iii) the first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof is a primer pair selected from the group consisting of the pair SEQ ID NO: 13 and 14, the pair SEQ ID NO: 15 and 16, and the pair SEQ ID NO: 17 and 18; and/or
(iv) the first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof is a primer pair selected from the group consisting of the pair SEQ ID NO: 19 and 20, the pair SEQ ID NO: 21 and 22, and the pair SEQ ID NO: 23 and 24.
8. The method according to any one of claims 1 to 7, wherein the differentiation of iPSCs in step a) is effected via directed differentiation.
9. The method according to any one of claims 1 to 8, wherein the samples from the iPSCs, the endodermal cells, the ectodermal cells, and the mesodermal cells of step b) are cDNA samples, preferably obtained by reverse transcription reaction from RIMA isolated from the respective cells.
10. The method according to any one of claims 1 to 9, wherein the nucleotide amplification reactions of step d) are quantitative polymerase chain reactions (qPCR) or digital polymerase chain reactions (dPCR).
11. The method according to any one of claims 1 to 10, wherein the score between 3 and 4 in step e) indicates integrity of the iPSCs.
12. Kit for determining the integrity of iPSCs, comprising a) a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof; b) a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof; c) a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof; d) a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof.
13. In vitro method for determining the status of differentiated iPSCs comprising the following steps: a) providing a differentiated iPSCs-derived sample; bl) mixing a first aliquot of the differentiated iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, thereby forming an amplification mixture; c) performing nucleotide amplification reaction of the amplification mixture and quantifying the amplification products; d) analysing the results of quantification of the amplification products, thereby determining the status of the differentiated iPSCs.
14. The method of claim 13, wherein the method additionally comprises at least one of the following steps: b2) mixing a second aliquot of the differentiated iPSCs-derived sample with a second set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, thereby forming an amplification mixture; b3) mixing a third aliquot of the differentiated iPSCs-derived sample with a third set of amplification primers capable of amplifying the gene specific for either endodermal, ectodermal, or mesodermal cells, or a part thereof, thereby forming an amplification mixture.
15. In vitro method for determining the status, especially the differentiation status, of iPSCs comprising the following steps: a) providing an iPSCs-derived sample; bla) mixing a first aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2a) mixing a second aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3a) mixing a third aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4a) mixing a fourth aliquot of the iPSCs-derived sample with a first set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture; c) performing nucleotide amplification reactions of the amplification mixtures and quantifying the resulting respective amplification products; d) analysing the results of the quantification of the amplification products in step c), thereby determining the status of the iPSCs.
16. The method according to claim 15, wherein the method additionally comprises at least one of the following steps: bib) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4b) mixing a further aliquot of the iPSCs-derived sample with a second set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
17. The method according to claim 16, wherein the method additionally comprises at least one of the following steps: blc) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for pluripotent cells or a part thereof, thereby forming an amplification mixture; b2c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for endodermal cells or a part thereof, thereby forming an amplification mixture; b3c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for ectodermal cells or a part thereof, thereby forming an amplification mixture; b4c) mixing a further aliquot of the iPSCs-derived sample with a third set of amplification primers capable of amplifying a gene specific for mesodermal cells or a part thereof, thereby forming an amplification mixture.
18. Computer-implemented method for determining the quality of an induced pluripotent stem cell (iPSC)-derived sample, the method comprising the following steps: providing an iPSC-derived sample to be analysed; performing a PCR. analysis on the iPSC-derived sample and obtaining fluorescence signal-derived values for n cycles of the PCR.; determining a quality parameter for the iPSC-derived sample using a trained classifier and the obtained fluorescence-signal- derived values.
19. Method according to claim 18, characterised by the following: providing a data set comprising fluorescence signal-derived values and classification data, wherein the classification data assigns a quality parameter to each of the fluorescence signal- derived values; splitting the data set into a first group of data comprising N pairs of fluorescence signal-derived values and corresponding quality parameters, wherein the first group comprises pl percent of the entire data set, and a second group of data comprising M pairs of fluorescence signal-derived values and corresponding quality parameters, wherein the second group comprises p2 percent of the entire data set, the sum of pl and p2 being 100; using the first group of data for training c classifiers and using the second group of data for testing the c classifiers, obtaining a rating parameter n for each of the c classifiers, wherein each rating parameter n indicates the performance of each of the c classifiers; and selecting from the c classifiers the classifier with the highest rating for analysing the iPSC-derived sample to be analysed.
20. Computer-readable storage medium comprising instructions which, when executed by a computer, cause the computer to carry out the method of claim 18 or 19.
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