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WO2023118050A1 - Utilisation de nouveaux marqueurs pour détecter des cellules souches pluripotentes - Google Patents

Utilisation de nouveaux marqueurs pour détecter des cellules souches pluripotentes Download PDF

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WO2023118050A1
WO2023118050A1 PCT/EP2022/086855 EP2022086855W WO2023118050A1 WO 2023118050 A1 WO2023118050 A1 WO 2023118050A1 EP 2022086855 W EP2022086855 W EP 2022086855W WO 2023118050 A1 WO2023118050 A1 WO 2023118050A1
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cells
cell
cell population
stem cells
expression
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Jonathan NICLIS
Josefine Rågård CHRISTIANSEN
Fabian Vinzenz ROSKE
Paschalis EFSTATHOPOULOS
Hanni WILLENBROCK
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Novo Nordisk AS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to the field of stem cells, such as human embryonic stem cells.
  • Methods are provided for detecting pluripotent stem cells (PSCs) in an in vitro cell population of differentiated cells derived from PSCs.
  • PSCs pluripotent stem cells
  • the stem cell products for such treatment may be derived from human pluripotent stem cells (hPSCs) such as but not limited to embryonic stem cells or induced PSCs.
  • hPSCs human pluripotent stem cells
  • Human PSCs are largely undifferentiated cells with the potential to proliferate and differentiate into a number of more specialized cells of the human body.
  • Established methods for obtaining stem cell-derived differentiated cells for the treatment of various conditions have already been developed, including protocols for providing ventral midbrain neural cells, retinal pigment epithelium (RPE) cells, neural retina cells, pancreatic islets containing beta cells, and cardiomyocytes.
  • RPE retinal pigment epithelium
  • Such protocols are typically not completely efficient and often result in a cell population comprising the intended cells as well as other cell types that may or may not be suitable of use in a final medicinal product. Furthermore, for some treatments it may not be viable to administer the fully differentiated or matured cells. In these cases, the differentiation of the cells is not fully completed in vitro as the cells are then intended to further mature in vivo after administration into the patient. Depending on the level of maturity, the medicinal product may still contain some small fraction of cells in a mitotic stage with high capacity to proliferate.
  • a stem cell-derived population wherein the differentiated cells have not fully matured may comprise a mixture of cells at various developmental stages. Even for cell populations derived according to a differentiation protocol for which fully matured cells are intended a subset of the cells may still be at a mitotic stage or may even be pluripotent.
  • a stem cell- derived product for administration comprises PSCs and/or PSC-like cells with the inherent potential to proliferate and develop into almost any cell type.
  • the major concern being the risk of uncontrollable proliferation of the cells, which could potentially develop into a teratoma or malignant tumor or a cancer-like state.
  • Continued development of the differentiation protocols as well as optional purification processes may result in a highly pure cell population.
  • Even still, to ensure patient safety and to comply with regulations by health authorities a quality control of stem cell- derived products is required for verifying that a product is not contaminated with residual undifferentiated cells, in particular PSCs or PSC-like cells.
  • pluripotent markers are well characterized in human PSCs. As PSCs are differentiated into a specific germ layer and further into a more specialized cell type the gene expression of the cell will change. This suggests for using genetic markers to establish the type and maturity of the cell. Multiple markers identifying human PSCs are known. Depending on the cell type into which a PSC is differentiated many markers expressed at the pluripotent stage will to some extent downregulate. This can be utilized in identifying PSCs in a cell population of differentiated cells. However, the timing and extent of the expression of pluripotent markers being down regulated differs for different cell types making a generic method of detecting the PSCs difficult. Furthermore, it is known that well-established markers for pluripotency such as S0X2, NANOG, PODXL, CD9 and LIN28A may also be expressed in some cells that have differentiated and lost pluripotency.
  • pluripotency such as S0X2, NANOG, PODXL, CD9 and LIN28A may also be expressed
  • a method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in the cell population, wherein the marker is selected from UNC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the present inventors have found that the expression of these particular markers are downregulated as the stem cells lose pluripotency.
  • a cell population comprising differentiated cells derived from PSCs, wherein the cell population is devoid of cells expressing one or more markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • Figure 1 shows combined tSNE plot of single cell sequencing results from several different cell types, every dot represents a single cell.
  • Clusters 1-4 are endoderm/pancreatic islet cells.
  • Clusters 5-7 and 18 are mesoderm/cardiomyocyte cells.
  • Cluster 8 is ectoderm/ retinal pigment epithelial cells.
  • Clusters 9-14 are undifferentiated/pluripotent stem cells.
  • Clusters 15-17 are mesoderm/mesenchymal stem cells.
  • Clusters 19-21 are ectoderm/ventral midbrain neural stem cells.
  • Figures 2-4 show gene expression distribution of cardinal lineage and cell type markers throughout multi-cell type combined dataset. Expression shown for the pluripotency gene POLI5F1 (Figure 2), retinal epithelial cell gene LHX2 ( Figure 3), and ventral midbrain neural stem cell gene EN-1 ( Figure 4).
  • Figures 5-7 show gene expression distribution of cardinal lineage and/or cell type markers throughout multi-cell type combined dataset. Expression shown for the cardiomyocyte gene NKX2.5 ( Figure 5), mesenchymal stem cell gene NT5E ( Figure 6), and pancreatic islets gene NKX2.2 ( Figure 7).
  • Figures 8-10 show gene expression distribution of cardinal undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the marker LIN28A ( Figure 8), NANOG ( Figure 9), and POLI5F1 ( Figure 10).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 11-13 show gene expression distribution of cardinal undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene PODXL ( Figure 11), SOX2 ( Figure 12), and CD9 ( Figure 13).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 14-17 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene DNMT3B ( Figure 14), LINC00678 ( Figure 15), USP44 ( Figure 16) and LINC00458 ( Figure 17).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 18-21 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene CNMD (Figure 18), POL3G ( Figure 19), AC009446.1 (Figure 20), and SCGB3A2 ( Figure 21).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 22-25 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene VRTN ( Figure 22), ZIC2 ( Figure 23), CRABP1 ( Figure 24), and CYP2S1 ( Figure 25).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 26-29 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene ALPL ( Figure 26), AL353747.4 (Figure 27), VASH2 ( Figure 28), and TNNT1 ( Figure 29). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster. Figures 30-33 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset.
  • Figures 34-37 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene FOXH1 ( Figure 34), CLDN6 (Figure 35), ZFP42 ( Figure 36), and CLDN7 ( Figure 37).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 38-40 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene SFRP2 (Figure 38), HSALNG0067850 ( Figure 39), and AC104461.1 ( Figure 40).
  • A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.
  • Figures 41-44 show examples of real time qPCR results showing the mRNA expression of a selection of markers in undifferentiated hPSCs and in various differentiated cell types that represent the 3 germ layers. Expression shown for the gene SCGB3A2 ( Figure 41), CYP2S1 ( Figure 42), LINC00458 ( Figure 43), and L1TD1 ( Figure 44).
  • Figures 45-48 show examples of real time qPCR results showing the mRNA expression of a selection of markers in undifferentiated hPSCs and in various differentiated cell types that represent the 3 germ layers. Expression shown for the gene VRTN ( Figure 45), ZFP42 ( Figure 46), ALPL ( Figure 47), and LIN00678( Figure 48).
  • Figure 49 shows results of real time qPCR showing OCT4 (POLI5F1) and VRTN genes expression in D60 hESC-RPE spiked-in with different concentrations of E1C3, hES.
  • the data are represented as fold change compared to hES-RPEs after being normalised to ACTB expression.
  • the VRTN fold change curve shows better linearity up to 0.001% compared to the classic pluripotency gene OCT4 and can be used for detection of pluripotent cells in the RPE drug product with a LoD of 0.001%.
  • Figure 50 shows a histogram of mean copies/pl normalized to the input cDNA per sample.
  • the numbers on the x axis refer to the percentage of stem cells spiked in the surrogate RPE cells.
  • Note y axis is in logarithmic scale to allow visualization of output signal for each sample.
  • day 0 refers to the initiation of the protocol, this be by for example but not limited to plating the stem cells or transferring the stem cells to an incubator or contacting the stem cells in their current cell culture medium with a compound prior to transfer of the stem cells.
  • the initiation of the protocol will be by transferring undifferentiated stem cells to a different cell culture medium and/or container such as but not limited to by plating or incubating, and/or with the first contacting of the undifferentiated stem cells with a compound that affects the undifferentiated stem cells in such a way that a differentiation process is initiated.
  • pluripotent stem cell an undifferentiated cell having differentiation potency and proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency.
  • pluripotent stem cells undifferentiated stem cells or undifferentiated pluripotent stem cells may be used interchangeably.
  • the stem cell includes subpopulations such as PSC, multipotent stem cell, unipotent stem cell and the like according to the differentiation potency.
  • PSC refers to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to three germ layers (ectoderm, mesoderm, endoderm).
  • the multipotent stem cell means a stem cell having a potency to differentiate into plural types of tissues or cells, though not all kinds.
  • the unipotent stem cell means a stem cell having a potency to differentiate into a particular tissue or cell.
  • a PSC can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the PSC include embryonic stem cell (ES cell), EG cell (embryonic germ cell), induced pluripotent stem cell (iPSC) and the like.
  • Muse cell Multi-lineage differentiating stress enduring cell obtained from mesenchymal stem cell (MSC), and GS cell produced from reproductive cell (e.g., testis) are also encompassed in the PSC.
  • iPSCs are a type of PSC that can be generated directly from adult cells. By the introduction of products of specific sets of pluripotency-associated genes adult cells can be converted into PSCs.
  • Embryonic stem cells can be produced by culturing cells from a blastomere or the inner cell mass of a blastocyst. Such cells can be obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available.
  • cells before and after the purification step will be referred to as cell drug substance (DS) and cell drug product (DP), respectively.
  • a method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in the cell population, wherein the marker is selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the term “cell population” refers to a defined group of cells, which may be in vitro or in vivo. Typically, the group of cells will be isolated in vitro in a container. In a preferred embodiment, the method according to the present invention is carried out in vitro. In an embodiment, the in vitro container is a suitable substrate such as a microwell.
  • screening refers to the action of examining the cell population for the presence of one or more cells having a certain genotype or phenotype, such as pluripotency.
  • the genotype and phenotype may be established based on the expression of markers.
  • the term “marker” or “markers” refers to a naturally occurring identifiable expression made by a cell, which can be correlated with certain properties of the cell.
  • the marker is a genetic or proteomic expression, which can be detected and correlated with the identity of the cell.
  • the markers may be referred to by gene. This can readily be translated into the expression of the corresponding mRNA and proteins.
  • the term “expression” in reference to a marker refers to the lack or presence in the cell of a molecule, which can be detected.
  • the expressed molecule is mRNA or a protein.
  • the PSCs are detected, and optionally identified, on a transcriptomic and/or proteomic level.
  • the marker is the genetic expression of a gene, which can be correlated with pluripotency of a stem cell.
  • the expression of the marker may be detected at any suitable level, such as at mRNA or protein level.
  • a cell can be defined by the positive or negative expression of a marker, i.e. the properties and state of a cell may equally be correlated based on the expression of a certain marker as well as the lack thereof. When referring to specific markers the presence or lack of expression may be denoted with + (plus) or - (minus) signs, respectively.
  • the term “detecting” in reference to expression means measuring a signal to establish the presence of undifferentiated stem cells in a cell population. “Detecting” according to the method does not imply that a positive signal must be obtained, which would not be the case if the cell population does not comprise any undifferentiated stem cells. Any suitable signal may be used to establish the presence of PSCs, such as by the emission of light from e.g. fluorescent molecules. Numerous techniques are readily available to detect and optionally identify markers in a cell population. In one embodiment, the cell population is screened using bulk RNA-seq (RNA sequencing) analysis. As used herein, the term “bulk” when referring to screening means analyzing the expression of a marker in a cell population not the individual cells. As used herein, “DNMT3B” refers to the gene denoted DNA Methyltransferase 3 Beta.
  • LINC00678 refers to the gene denoted Long Intergenic Non-protein Coding RNA 678.
  • USP44 refers to the gene denoted Ubiquitin Specific Peptidase 44.
  • LINC00458 refers to the gene denoted Long Intergenic Non-protein Coding RNA 458.
  • CNMD refers to the Chondromodulin.
  • POLR3G refers to the gene denoted RNA polymerase III subunit G.
  • AC009446.1 refers to the novel transcript also known as ENSG00000254277.
  • SCGB3A2 refers to the gene denoted Secretoglobin family 3A member 2.
  • VRTN refers to the gene denoted Vertebrae development associated.
  • ZIC2 refers to the gene denoted Zic family member 2.
  • CRABPT refers to the gene denoted Cellular Retinoic Acid Binding Protein 1.
  • CYP2S1 refers to the gene denoted Cytochrome P450 family 2 Subfamily S member 1.
  • APL refers to the gene denoted Alkaline Phosphatase, biomineralization associated.
  • AL353747.4 refers to the novel transcript also known as ENSG00000280707.
  • VASH2 refers to the gene denoted Vasohibin 2.
  • TN NT1 refers to the gene denoted Troponin T1, slow skeletal type.
  • L1TD1 refers to the gene denoted LINE1 type transposase domain containing 1.
  • GAL refers to the gene denoted Galanin and GMAP prepropeptide.
  • SFRP2 refers to the gene denoted Secreted Frizzled Related Protein 2.
  • DPPA4 refers to the gene denoted Developmental Pluripotency Associated 4.
  • TDGF1 refers to the gene denoted teratocarcinoma-derived growth factor 1.
  • FOXHT refers to the gene denoted Forkhead box H1.
  • Claudin 6 refers to the gene denoted Claudin 6.
  • ZFP42 refers to the gene denoted ZFP42 zinc finger protein.
  • Claudin 7 refers to the gene denoted Claudin 7.
  • AC104461.1 refers to the novel transcript also known as ENSG00000230623.
  • AC0064802.1 refers to the novel transcript also known as ENSG00000254339.
  • the expression of one or more markers selected from DNMT3B, LINC00678, USP44, CNMD, SFRP2, DPPA4, and TDGF1 is further detected.
  • further detected is meant that the expression of one or more markers is detected in a cell population in addition to the detection of the expression of other markers.
  • the presence of undifferentiated stem cells in a cell population is established by the positive expression of either one of the markers using bulk analysis of the cell population.
  • the bulk analysis is by RNA-seq analysis.
  • a cell population or its supernatant is screened for a secreted product of markers according to the present invention.
  • the cell population comprises differentiated cells derived from PSCs.
  • differentiated cells in respect to stem cells refers to PSCs, which have undergone a process wherein the cells have progressed from an undifferentiated state to a specific differentiated state, i.e. from an immature state to a less immature state or to a mature state. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has matured, partially differentiated or fully differentiated. “Differentiated cells” are therefore considered to be cells which have previously been classified as PSCs but allowed to differentiate into the cell type of a certain germ layer.
  • the method comprises an initial step of differentiating PSCs into a cell population of differentiated cells derived from the PSCs.
  • differentiated refers to subjecting the PSCs to a method which progresses the cells from an undifferentiated state to a differentiated state.
  • a step of differentiating PSCs involves culturing the cells under certain conditions and/or contacting the cells with certain factors.
  • the PSCs are human PSCs. In a further embodiment, the PSCs are human embryonic stem cells. In another embodiment, the PSCs are induced pluripotent stem cells.
  • the differentiated cells are selected from ventral midbrain neural stem cells, forebrain neural cells, spinal cord neural stem cells, retinal pigment epithelium (RPE) cells, pancreatic islets (containing beta cells), mesenchymal stem cells, macrophages cardiomyocytes or other differentiated cells that are not undifferentiated stem cells.
  • RPE retinal pigment epithelium
  • pancreatic islets containing beta cells
  • mesenchymal stem cells containing macrophages cardiomyocytes or other differentiated cells that are not undifferentiated stem cells.
  • a protocol for obtaining ventral midbrain neural stem cells is disclosed in patent application WO 2016/162747.
  • ventral midbrain neural stem cells may express of one or more of the markers F0XA2, LMX1B, 0TX2, EN1, PITX3, and TH.
  • a protocol for obtaining RPE cells is disclosed by Osakada et al (J Cell Sci. 2009 Sep 1 ;122(Pt 17):3169-79. doi: 10.1242/jcs.050393. Epub 2009 Aug 11. “In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction”) or by Kuroda et al. (Stem Cell Res. 2019 Aug;39:101514. doi: 10.1016/j.scr.2019.101514. Epub 2019 Jul 25.
  • Beta cells may be defined by the expression of the markers NKX6.1+/INS+/GCG-.
  • a protocol for obtaining cardiomyocytes is disclosed by Yap et al. (Cell Rep. 2019 Mar 19;26(12):3231- 3245. e9. doi: 10.1016/j.celrep.2019.02.083. “In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors”) or by Fernandes et al. (Stem Cell Reports. 2015 Nov 10; 5(5): 753-762. doi: 10.1016/j.stemcr.2015.09.011. “Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair”).
  • the present inventors analyzed cell populations of RPE cells, ventral midbrain neural stem cells, pancreatic islets containing beta cells, mesenchymal stem cells and cardiomyocytes, respectively, using single cell RNA-seq. None of the cell populations contained cells expressing the markers DNMT3B, LINC00678, USP44, LINC00458, CNMD, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, SFRP2, DPPA4, TDGF1, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the cell population is in vitro. Most commonly, the cell population for screening will be an in vitro stem cell-derived product of differentiated cells intended for therapy. In one embodiment, the cell population is provided from a biopsy. Such biopsy may be obtained directly from a patient and analyzed in vitro to screen for PSCs.
  • the method as disclosed herein is carried out in vitro.
  • the cell population is derived in vitro.
  • the method is carried out on an in vitro stem cell-derived cell culture, which has not been directly taken from a human or animal body. Accordingly, in an embodiment the cell population is not provided from a biopsy.
  • the method comprises the step of identifying PSCs or PSC-like cells in the cell population.
  • PSC-like cells means cells that have lost pluripotency but are still sharing some characteristics with PSCs such as some gene expression, capacity to proliferate or any other feature similar to PSCs.
  • identifying is meant establishing or indicating a strong link between detecting the expression of certain markers in a cell population and a specific cell of that cell population.
  • PSCs or PSC-like cells are detected, and optionally identified, by single cell sequencing.
  • the cell population is screened using fluorescence-activated cell sorting (FACS).
  • a cell population comprising differentiated cells derived from PSCs, wherein the cell population is devoid of cells expressing one or more of the marker selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the marker selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the cell population screened contains a PSC, it has a limit of detection (LoD) value of the expression of one or more of the markers LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1 below 0.1 , 0.01 , 0.001 , 0.0001 or 0.00001 % of hPSC mixed in the differentiated cells compared to a spike-in reference cell population.
  • LiD limit of detection
  • the cell population has a limit of detection value of the expression of one or more of the markers below 0.1, 0.01 , or 0.001 % of hPSCs mixed in the differentiated cells compared to a spike-in reference cell population.
  • the term “devoid” is defined by the negative detection of one or more of the expression markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the cell population has been screened according to the method of the first aspect of the present invention.
  • a method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in a cell population, wherein the marker is selected from LINC00458, POLR3G, AC009446. 1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.
  • the cell population comprises differentiated cells derived from Pluripotent Stem Cells (PSCs).
  • PSCs Pluripotent Stem Cells
  • the differentiated cells are selected from ventral midbrain neural stem cells, forebrain neural cells, spinal cord neural stem cells, retinal pigment epithelium (RPE) cells, pancreatic islets, mesenchymal stem cells, macrophages and cardiomyocytes.
  • RPE retinal pigment epithelium
  • cDNA complementary DNA, DNA synthesized from a single-stranded RNA
  • RT-PCR qPCR
  • ddPCR ddPCR
  • a method of screening a cell population comprising dopaminergic progenitor cells for undifferentiated stem cells comprising the step of detecting the expression of the marker ZFP42.
  • a cell population comprising differentiated cells derived from PSCs, wherein the population is devoid of cells expressing one or more markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1 , CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, FOXH1, CLDN6, CLDN7, ZFP42, AC104461.1 , AC0064802.1, and AC0064802.7.
  • markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1 , CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, FOXH1, CLDN6, CLDN7, ZFP42, AC104461.1 , AC0064802.1, and AC0064802.7.
  • Example 1 RNA sequencing methodology and experimental design to identify markers unique to undifferentiated PSCs
  • hPSC samples and differentiated cell types were processed with single cell RNA sequencing in order to compare the transcriptomic signature for the purpose of identifying, in an unbiased manner, genes unique to or upregulated in undifferentiated hPSCs.
  • hPSCs samples were analysed. Firstly, three independent genetically distinct hPSC cell lines, one of which was a human induced pluripotent stem cell line and two of which were human embryonic stem cell lines were analysed. Further, three different culture conditions were selected that covered several different commercial medias (including mTeSR, iPSC-Brew and NutriStem) and different matrices including human laminins.
  • RNA sequencing To perform single cell RNA sequencing (scRNA-seq), cell clusters of undifferentiated PSCs as well as those of differentiated cells were dissociated into single cell suspensions with accutase, tryple select or other such reagents and 3000-10000 cells were processed using the 10X Genomics Chromium Platform and sequenced on a NextSeq550. Data was processed using 10X cellranger and the Seurat analysis package in R programming language. Samples were analysed, filtered for low quality or multiplet cells and analyzed separately for each individual experiment before combining the cells of the selected differentiated cell lineages of choice as well as the hPSCs into one dataset that were then analysed using the standard Seurat workflow as outlined for Seurat version 3, i.e. normalizing using SCTransform and finally using the first 29 principal components for the unified tSNE plot (Fig.1).
  • Example 2 Methods of generating differentiated cells of all three germ layers from hPSCs and positions in combined dataset
  • Differentiated cells derived from hPSCs were obtained according to several published protocols that generate cells that cover all three major germ layers, these are represented in clusters 1-8 and 15-21 (Fig. 1). Differentiation was performed to two ectoderm lineages, one to ventral midbrain neural stem cells following the Nolbrant et al 2017 (Nat Protoc. 2017 Sep;12(9):1962-1979.doi: 10.1038/nprot.2017.078. Epub 2017 Aug 31.’’Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation”) methodology and this sample is contained in clusters 19-21 (Fig.1). A second ectodermal lineage of the forebrain was differentiated following the Plaza Reyes et al 2020 protocol that produces retinal epithelium cells and corresponded to cluster 8 (Fig.1 ).
  • cardinal genes typically transcription factors
  • POLI5F1 the pluripotency transcription factor
  • LHX2 the cardinal retinal lineage transcription factor
  • EN-1 the cardinal midbrain transcription factor
  • the cardinal cardiac lineage transcription NKX2.5 was principally expressed in cardiomyocyte cell clusters 5-7 and 18 (Fig.5), while the cardinal mesenchymal plasma membrane marker NT5E was expressed in the mesenchymal stem cell population.
  • the pancreatic endoderm marker NKX2.2 (Doyle et al., 2007) was expressed in pancreatic islet clusters 1-4 (Fig.7).
  • Example 3 Computational comparison between hPSC and differentiated cell samples: Computational comparisons can be made between any cell clusters. To identify novel genes highly enriched or exclusively expressed in undifferentiated cells, the clusters of hPSCs (9-14; Fig.1 ) were compared to all differentiated cell clusters (clusters 1-8, 15-21; Fig.1 ). Highly enriched or exclusively expressed genes in undifferentiated cells were defined using the Wilcoxon Rank Sum test for differential gene expression, while only considering genes with a foldchange of minimum 0.25 between the 2 groups and where a gene is expressed in at least 25% of the hPSC cells.
  • cardinal pluripotency genes including POLI5F1 , LIN28A, SOX2, PODXL, CD9 and NANOG (Yuin-Han Loh et al., 2006) as shown in Table 1 (results in bold numbered 1, 12, 32, 33, 41 and 66 respectively) confirming the validity of our method for identifying markers associated with undifferentiated pluripotent stem cells.
  • the expression of cardinal pluripotency markers was checked in our combined dataset and in all cases genes were expressed throughout pluripotency cell clusters (Fig.8-10 and 11-13). However, many of these cardinal pluripotency markers were found also to be expressed in differentiated cell clusters and were not confined to hPSC cells, e.g.
  • LIN28A was expressed in 2 different differentiated cell types and 22,8% of all differentiated cells (Fig.8). Many other cardinal pluripotency markers were found not to be expressed by all undifferentiated cells, e.g. NANOG which was not expressed in 18,8% of undifferentiated cells (Fig 9). Surprisingly, only one of the cardinal pluripotency markers, POLI5F1 , appeared to uniquely identify all pluripotent cells (Fig.10) and thus would avoid the risk of false positive or negative results when used to screen a population of cells for the presence of undifferentiated cells (for which our mixed cell dataset is a theoretical example of).
  • Example 4 Identification of universal markers of undifferentiated pluripotent stem cells.
  • Example 5 Use of Quantitative real-time PCR (qRT-PCR) to compare expression levels in pure hPSC and differentiated cell populations
  • qPCR In the traditional RT-PCR (or qPCR for simplification), the amplification of a sequence is followed by emerging fluorescence during the PCR reaction (Higuchi et al., Biotechnology (N Y). 1992 Apr;10(4):413-7. doi:10.1038/nbt0492-413). qPCR is usually conducted to compare relative amounts of a target sequence between samples. This technique monitors the amplification of the target in real-time via a target-specific fluorescent signal emitted during amplification. In qPCR, the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels.
  • Ct threshold cycle
  • Cp crossing point
  • the level of expression of the gene in hPSCs vs the levels of expression in the cell type of interest is the fold change difference which indicates how good is this marker for detecting a potential hPSC contaminant in the respective drug product.
  • a fold change over 1000 times is expected to give a good sensitivity for detection of PSCs using an RNA based assay e.g. VRTN for RPEs and ZFP42 for vmDA neurons.
  • Example 6 Use of a novel universal marker to identify residual hPSCs in a mixed population, at a lower level of detection than the most robust traditional marker (POU5F1)
  • the fold change relative to RPE expression was calculated using the ddCt (delat- delta Ct) method and GAPDH expression (housekeeping gene) as an endogenous control. Briefly, for each sample the dCt was initially calculated by subtracting the Ct value for the gene of interest from the Ct value of GAPDH. Subsequently, since we wanted to normalize everything to the expression level of the RPE cells, the dCt of the RPE sample for each gene was subtracted from the dCt of each of the other samples in order to calculate the ddCt. Finally, the fold change was calculated using the formula 2 A (-ddCt).
  • Example 7 Use of the novel universal marker ZFP42 to identify residual hPSCs in a mixed population of vmDA neurons, with high sensitivity
  • Droplet digital PCR offers absolute quantification of nucleic acid targets by counting discrete water-in-oil droplets encapsulating nucleic acid molecules.
  • the massive sample partitioning into -20000 droplets effectively enriches rare templates and thus provides a sensitive detection of rare sequences in a high background.
  • the PCR amplification is carried out within each droplet, and the target concentration is calculated based on the number of positive and negative droplets.
  • Positive droplets contain at least one copy of the target molecule, and thus exhibit a higher fluorescent signal compared to the negative droplets.
  • the fraction of positive droplets is then fitted into a Poisson distribution-based algorithm by the QX manager standard Edition software version 1.2 or newer.
  • the readout is the target concentration in units of copies/pl, which can be used for downstream calculations to determine copies/cell in the overall population. This value, together with the evaluation of stem cells, can be used to give an estimate of the presence and potentially also the fraction of pluripotent cells in the sample.

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Abstract

La présente invention concerne un procédé de criblage d'une population cellulaire à la recherche de cellules souches indifférenciées en détectant l'expression d'un ou plusieurs marqueurs dans la population cellulaire, dont l'expression est effectivement réduite au fur et à mesure que les CSP se différencient en cellules spécialisées de l'une ou l'autre des trois couches germinales.
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CN119979719B (zh) * 2025-02-26 2026-01-13 中国食品药品检定研究院 一种基于基因的多能干细胞残留通用型检测方法

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