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WO2025114490A1 - Méthode de reprogrammation de cellules - Google Patents

Méthode de reprogrammation de cellules Download PDF

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Publication number
WO2025114490A1
WO2025114490A1 PCT/EP2024/084006 EP2024084006W WO2025114490A1 WO 2025114490 A1 WO2025114490 A1 WO 2025114490A1 EP 2024084006 W EP2024084006 W EP 2024084006W WO 2025114490 A1 WO2025114490 A1 WO 2025114490A1
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Prior art keywords
cell
tfs
cells
combinations
reprogramming
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Yen Choo
Chiara NADEO
Harshyaa MAKHIJA
Giuseppe D'AGOSTINO
Marina Tarunina
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Plasticell Ltd
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Plasticell Ltd
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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
    • G01N33/5005—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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks

Definitions

  • Cell therapy the treatment of patients with severe diseases using cells expanded and potentially modified in the laboratory, is a very promising technology that is delivering excellent results in oncology (both for leukemias and solid tumors) (Ottaviano et al. 2022) and regenerative medicine (for instance in the recovery of burn victims) (Cossu et al. 2018).
  • Cells can be safely derived from patients or donors, manipulated to expand, differentiate and potentially correct genetic defects or enhance their therapeutic capabilities, and then administered back to the patient.
  • ATMPs advanced therapy medicinal products
  • One of the major limitations is the lack of access to therapeutically useful amounts of cells.
  • stem cells for regenerative medicine consists in the unprecedented possibility to derive any type of cell from a human pluripotent stem cell such as an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESCs).
  • iPSC induced pluripotent stem cell
  • hESCs human embryonic stem cell
  • iPSC induced pluripotent stem cell
  • hESCs human embryonic stem cell
  • These pluripotent stem cells have the potential to generate in vitro any human tissue, since they are part - or very similar to - the embryonic structures that give rise to all primordial structures in our body.
  • the main challenge is directing these stem cells in robust and scalable ways to develop into specific cell types useful for therapy.
  • the expansion and/or differentiation of clinically useful cell products from donor-derived stem cells requires costly and time-consuming testing and optimisation of cell culture conditions that are tailored to the specific application and cell type of interest.
  • Cell culture conditions must be formulated by carefully considering the developmental cues in the human embryo, many of which are imperfectly understood. In any case, these may not be effective in the absence of the physiological niche which provides additional stimuli that are difficult to model in cell culture, e.g. specific cell-cell interactions, extracellular matrix composition and tissue stiffness. There is no a priori method to determine which of these conditions will yield a highly homogeneous, correctly differentiated cell population, with high reproducibility and in a process that is scalable and has the potential to meet regulatory requirements.
  • Cell reprogramming the process of converting one cell type to another cell type through manipulation of gene regulatory networks that define cell identity, may have great promise for regenerative medicine but has yet to be applied broadly.
  • the identification of factors to directly reprogram the identity of cell types is currently limited by, amongst other things, the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable.
  • TFs exogenous transcription factors
  • Yamanaka Yamanaka S. Cell 2006; 126:663-76; PMID: 16904174
  • a small set of TFs namely OCT4, KLF4, SOX2 and MYC (OKSM)
  • iPSC induced pluripotent stem cell
  • other groups have demonstrated that it is possible to convert fibroblasts to hepatocytes, to cardiomyocytes and to several other cell types (Huang P, et al..
  • iPSC pluripotent cell
  • forward programming where a less differentiated cell such as an iPSC is reprogrammed into a specific cell type (Dalby et al, Stem Cell Reports 11 :1462-1478)
  • direct conversion where one differentiated cell type is switched into another without going through a pluripotent or progenitor state (Zhou et al, Nature 455:627-632); as well as combinations of the foregoing such as (iii) ‘indirect (or primed) reprogramming’ where ectopic OKSM is expressed transiently to effect partial reprogramming to a less differentiated (but not fully pluripotent) intermediate, followed by forward programming to obtain a more differentiated cell type that differs from the starting cell type (Efe et al, Nat Cell Biol 13:215-222).
  • cell reprogramming differs qualitatively from traditional ‘cell differentiation’, both in biological mechanism and by the practical method by which it is achieved.
  • Cell differentiation occurs by the progressive restriction of developmental potential until a terminal state is reached - famously illustrated by Waddington who imagined a ball rolling from the top of a mountain towards a lower energy resting place in the valley below (The Strategy of the Genes, pub. Allen & Unwin, London, 1957) - whereas in cell reprogramming this is not necessarily true (and in the case of iPSCs it is precisely the opposite).
  • stem in culture are exposed to developmental cues such as morphogens, growth factors, hormones, small molecules etc.
  • the cell fate switch is directed by ectopic expression of transcription factors (TFs) which translocate to the cell nucleus and directly control gene expression, typically by binding gene regulatory elements in enhancer and promoter DNA and stimulating (or inhibiting) the transcription of mRNA.
  • TFs transcription factors
  • the TFs that orchestrate cell reprogramming may themselves regulate one or more TFs amongst other genes, inducing and/or stabilising a Gene Regulatory Network (GRN) that specifies cell fate.
  • GRN Gene Regulatory Network
  • One of the best understood GRNs is the pluripotency gene regulatory network (PGRN) that is induced and stabilised by adding the Yamanaka factors OKSM to a (differentiated) cell.
  • PGRN pluripotency gene regulatory network
  • OCT4 triumvirate of pluripotency-inducing TFs
  • SOX2 SOX2
  • NANOG triumvirate of pluripotency-inducing TFs
  • These TFs regulate a larger network of interconnected secondary genes.
  • the core TFs do so in a cooperative manner, and form autoregulatory and feed-forward gene circuits that result in network stability.
  • NANOG and LIN28 can replace KLF4 and MYO in the Yamanaka cocktail, indicating a specific GRN can be induced by alternative means.
  • reprogramming can also be affected by non-coding RNAs that regulate gene expression, such as LincRoR and Let7 that affect key nodes in the PGRN, therefore in the present application non-coding RNAs that regulate gene expression are included in the definition of TFs.
  • GRNs are essentially composed of genomic components such as genes and their cis- regulatory modules at the nodes, and of regulatory state components, i.e. TFs (including non-coding RNAs) which provide regulatory input into these modules.
  • TFs regulatory state components
  • the conventional method of determining reprogramming cocktails is to screen candidate TFs in a large pool (or a set of smaller pools) and the minimal core TFs then identified through a process of eliminating each TF in turn, as is exemplified in Takahashi and Yamanaka’s (Cell 126:663-676) seminal experiment in which pools of 24 TFs were screened in a cell-based assay to identify OKSM in reprogramming fibroblasts to iPSCs.
  • Zhou et al (Nature 455:627-633) introduced a pool of 9 TFs into mouse pancreas in vivo, and observed pancreatic exocrine cells were reprogrammed into insulin-expressing beta cells.
  • Zhou et al were able to deduce, again by a process of elimination, that the effective reprogramming cocktail comprises PDX1 , NGN3 and MAFA.
  • these pooled screening experiments were unable to discover functional combinations of TFs beyond the minimal core cocktail, since the presence of the core TFs in a pool will mask the effect of eliminating other productive TF cocktails from the pool.
  • the present invention provides a method for determining the conditions required for reprogramming of a first cell type to a second cell type, by screening TF combinations in which TFs are added to the cells sequentially.
  • the invention provides a method for determining the conditions required for reprogramming of a first cell type to a second cell type, comprising the steps of: exposing a cell of a first cell type to a first TF; removing the first TF and exposing the cell to a different TF; optionally, repeating the foregoing step; identifying one or more cells which displays one or more markers of a second cell type and determining the identity and sequence of TFs to which the cell has been exposed.
  • W02004/031369 The split/pool techniques described in W02004/031369 are adaptable to sequential screening of the effects of the exposure of cells to multiple individual TFs and combinations of TFs.
  • Cell units in W02004/031369 are envisaged therein as beads comprising multiple cells which can be sorted together; in the present invention, it is envisaged that cell units such as those employed in W02004/031369 may be used, or single cells may be used and individually sorted.
  • a test unit may comprise multiple cells or a single cell.
  • the invention comprises the steps of: a. separately exposing a first plurality of test units each comprising a cell to different TFs or combinations of TFs and labelling the cells to indicate exposure to said TFs or combinations of TFs; b. pooling the plurality of test units, and subdividing the pool of test units to form a second plurality of test units; c. separately exposing the second plurality of test units to different TFs or combinations of TFs; d. optionally iteratively repeating steps (a) to (c) as required; e. identifying the cell type of the cells in the test units and deconvoluting the labels to identify the identity and sequence of TFs to which the cells have been exposed; f.
  • step (e) optionally, using the data from step (e) to inform the choice of TFs used in step (a), and repeating steps (a) to (e) as required; g. identifying a cell of the second cell type, and deconvoluting the labels to thereby identify the identity and sequence of TFs required to reprogram a cell of the first cell type into a call of the second cell type.
  • the present invention employs the approach described in W02004031369 (see Fig. 1).
  • that approach known as CombiCult®, a serial splitting and pooling of cells is used to expose multiple cells to many different reagent combinations.
  • the TFs used in step (a) and/or step (c) can be chosen on the basis of computational analysis of known cell reprogramming events.
  • the data used for analysis can be based on previous performance of the method of the invention, or derived from the prior art.
  • the computational selection is combined with the experimental selection provided by the split/pool selection methodology to permit unprecedented precision in determining both the identity of the factors required for cell reprogramming, and the ideal timing of their administration.
  • test units in the invention can take the form of beads, capsules, aggregates or the like comprising one or more cells. In some embodiments, distribution of cells to achieve an average of one cell per unit may result in some units being devoid of cells. In certain embodiments, the test unit is a single cell.
  • Labelling of test units can be performed in a variety of ways, for example as described in W02004/031369, including nucleic acid labels, radiofrequency encoded tags, fluorescent or optical tags, and spatial encoding of test units on a surface or matrix.
  • labelling is performed by modifying the nucleic acid of each cell, which allows its exposure to TFs to be tracked precisely.
  • Nucleic acid can be modified, for example, by retrovirus integration or genome editing techniques.
  • Test units comprising multiple or single cells, permits the cells to be exposed to an array of different TFs and combinations of TFs at different times, and for both the nature of the TFs and the teeing of exposure to be monitored.
  • the split/pool protocol comprises the following steps: (a) providing a first set of groups of test units each comprising one or more cells, and exposing said groups to desired TFs or combinations of TFs;
  • a pool may be exposed to a combination of four TFs in a first culture, and then split. Different splits of the pool may be exposed to different combinations of TFs, which may or may not comprise any of the TFs used in the first culture. After pooling and resplitting, further different splits may be exposed to yet further combinations of TFs which may comprise TFs from the first culture or the second culture, as well as novel TFs. Thus, cells in different splits are exposed to different TFs, as well as to the same TFs at different times.
  • the TFs are added individually to cell units in sequential method steps.
  • sequential method steps in a procedure may comprise either the addition of single TFs or the addition of combinations of TFs; alternatively sequential method steps in a procedure only comprise the addition of individual TFs.
  • a procedure may combine method steps which add groups of TFs with steps which add individual TFs, or may only add individual TFs.
  • a ’’procedure represents the performance of an entire method as represented by steps (a) to (f) above.
  • TFs may be added sequentially and individually, or in defined groups, in a large number of combinations, as explained above. This prevents the masking of the effect of a TF by dominant TFs which may be present in polled combinations of TFs used in conventional experiments.
  • the TFs are individually added sequentially to cells, such that the effect of each TF can be assessed in isolation.
  • the method of the invention allows thousands or millions of TFs and combinations of TFs to be tested, in a multiplexed high-throughput assay, to determine the conditions necessary to achieve the desired result with respect to any cellular process.
  • the method of the invention allows testing of sequential addition of TFs and therefore the determination of the action of each TF, when added in a plurality of sequential combinations, as well as the testing of multiple combinations amongst groups TFs. This permits the elucidation of the separate steps in a pathway of conversion from one cell type to a second sell type, rather than merely the overall conversion.
  • the invention provides a method for identifying a gene which influences a cellular process, comprising the steps of: a) determining the effect of one or more TFs or combinations of TFs on a test unit, in accordance with the foregoing aspect of the invention; b) analysing gene expression in said cell units when exposed to said TFs; and c) identifying genes which are differentially expressed under desired culture conditions.
  • the TFs used cause a change in the cellular process; these TFs are selected for the production of cells in which gene expression is analysed.
  • Gene expression may conveniently be analysed using any comparative expression monitoring technology including PCR-based techniques such as RT-PCR, Serial Analysis of Gene Expression (SAGE), RNA sequencing (RNA seq) including single-cell RNA seq, in-situ hybridisation including single-cell hybridisation, or array technology such as is widely available from suppliers such as Affymetrix.
  • the invention provides a method for producing a nucleic acid which encodes a gene product which influences a cellular process, comprising identifying a gene as above, and producing at least the coding region of said gene by nucleic acid synthesis or biological replication.
  • a method for inducing a cellular process in a cell comprising the steps of: a) identifying one or more genes which are differentially expressed in association with a cellular process in accordance with the invention; and b) modulating the expression of said one or more genes in the cell.
  • the expression of the genes in the cell can be modulated by, for example, transfecting or otherwise transferring the gene into the cell such that it is overexpressed in a transient or permanent manner.
  • the expression of the endogenous gene may be altered, such as by targeted enhancer insertion or the administration of exogenous agents which cause an increase (e.g. gratuitous inducers) or decrease (e.g.
  • the invention provides a method for identifying the state of a cellular process in a cell, comprising the steps of: a) identifying one or more genes which are differentially expressed in association with a cellular process as set forth above; and b) detecting the modulation of expression of said one or more genes in a cell, thereby determining the state of the cellular process in said cell.
  • the genes employed in this analysis encode cellular markers, which may be detected for instance by immunoassay.
  • the gene products may be enzymes that can be assayed for activity with fluorometric, colorimetric, radiometric, or other methodologies.
  • the invention further provides a method for regulating a cellular process, comprising the steps of: a) determining the effect of one or more TFs on a cell, in accordance with the foregoing aspect of the invention; b) exposing a cell to TFs which effect a change in the cellular process; and c) isolating the desired cell.
  • the invention provides for a method for producing a differentiated cell from a second, different differentiated cell type, in accordance with the foregoing aspect of the invention.
  • TFs identified in accordance with the invention may be expressed or synthesised by conventional chemical, biochemical or other techniques, and used in methods for regulating particular cellular processes in cells for example as described herein.
  • the invention relates to methods for developing and using a database of cell gene expression data correlated to exposure to TFs, which can provide improved TF selection in step (a) of the method of first aspect of the invention.
  • the database can be improved, further optimising TF selection and therefore the performance of the method of the invention.
  • Figure 1 is a schematic of the operation of the CombiCult® split/pool selection method.
  • Figure 2 is a schematic of the workflow for the CombiCult® method of the present invention.
  • Figure 3 is a schematic of genetic barcode labelling of cells in the method of the present invention.
  • Figure 4 illustrates the screening matrix for 10 transcription factors used in the generation of insulin-producing cells by the method of the invention.
  • Figure 5 is a photomicrograph showing insulin expression and cd49a expression in differentiated cells obtained by the method of the invention.
  • FIG. 6 Overview of negative and positive controls.
  • cPPs are placed onto the beads using the same media.
  • Media A consists of DMEM high glucose supplemented with Asc (50pg), 1 % B27, EGF (50ng), FGF-7 (50ng), and RA (50nM). The purpose here is to guide the differentiation process towards pancreatic progenitors, particularly considering the early stage of cPPs.
  • Media B is a combination of 5% KOSR, T3 (1 pM), and RA (25 nM), added to DMEM high glucose to foster endocrine progenitors.
  • Media C comprises DMEM low glucose enriched with 10% FBS, T3 (1 M), and Alk5i (10pM).
  • Figure 7 A is a graphical illustration of the combinations of transcription factors computationally analysed to determine the frequency of transcription factor addition to hits identified in Figure 5.
  • Figure 7B shows the results illustrated in Figure 7A in a bar graph.
  • Figure 8 illustrates selection of optimal transcription factors by the method of the invention, and also illustrates the deriving of timing information for transcription factor addition.
  • culture conditions refers to the environment which cells are placed in or are exposed to in order to promote growth or differentiation of said cells.
  • the term refers to the medium, temperature, atmospheric conditions, substrate, stirring conditions and the like which may affect the growth and/or differentiation of cells. More particularly, the term refers to specific reagents which may be incorporated into culture media and which may influence the growth and/or differentiation of cells.
  • the culture conditions are the reagent or combination of reagents to which a cell is exposed.
  • Cell A cell is defined as the smallest structural unit of an organism that is capable of independent functioning, or a single-celled organism, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable cell membrane or cell wall.
  • the cell may be prokaryotic, eukaryotic or archaebacterial.
  • the cell may be a eukaryotic cell.
  • Mammalian cells are preferred, especially human cells.
  • Cells may be natural or modified, such as by genetic manipulation or passaging in culture, to achieve desired properties.
  • a stem cell is defined in more detail below, and is a totipotent, pluripotent or multipotent cell capable of giving rise to more than one differentiated cell type.
  • Stem cells may be differentiated in vitro to give rise to differentiated cells, which may themselves be multipotent, or may be terminally differentiated.
  • Cells differentiated in vitro are cells which have been created artificially by exposing stem cells to one or more agents which promote cell differentiation.
  • Cellular process is any characteristic, function, process, event, cause or effect, intracellular or extracellular, which occurs or is observed or which can be attributed to a cell.
  • cellular processes include, but are not limited to, viability, senescence, death, pluripotency, morphology, signalling, binding, recognition, molecule production or destruction (degradation), mutation, protein folding, transcription, translation, catalysis, synaptic transmission, vesicular transport, organelle function, cell cycle, metabolism, proliferation, division, differentiation, phenotype, genotype, gene expression, or the control of these processes.
  • Cell unit A group of cells which may be a group of one. Pools of cell units may be sorted, subdivided and handled without substantially dissociating the cell units themselves, such that the cell unit behaves as a colony of cells and each cell in the cell unit is exposed to the same culture conditions.
  • a cell unit may comprise a bead to which is adhered a group of cells.
  • Totipotent A totipotent cell is a cell with the potential to differentiate into any type of somatic or germ cell found in the organism. Thus, any desired cell may be derived, by some means, from a totipotent cell.
  • Pluripotent A pluripotent cell is a cell which may differentiate into more than one, but not all, cell types.
  • Label A label or tag is a means to identify a cell unit and/or determine a culture condition, or a sequence of culture conditions, to which the cell unit has been exposed.
  • a label may be a group of labels, each added at a specific culturing step; or a label added at the beginning or the experiment which is modified according to, or tracked during, the culturing steps to which the cell unit is exposed; or simply a positional reference, which allows the culturing steps used to be deduced.
  • a label or tag may also be a device that reports or records the location or the identity of a cell unit at any one time, or assigns a unique identifier to the cell unit. Examples of labels or tags are molecules of unique sequence, structure or mass; or fluorescent molecules or objects such as beads; or radiofrequency and other transponders; or objects with unique markings or shapes.
  • a label is a genetic barcode, as described further herein.
  • a cell is exposed to culture conditions or reagents when it is placed in contact with a medium, or grown under conditions which affect one or more cellular process(es) such as the growth, differentiation, or metabolic state of the cell.
  • the culture conditions comprise culturing the cell in a medium in the presence of a reagent
  • the cell is placed in the medium with a reagent present for a sufficient period of time for it to have an effect.
  • the conditions are temperature conditions, the cells are cultured at the desired temperature.
  • pooling involves the admixture of the groups to create a single group or pool which comprises cell units of more than one background, that is, that have been exposed to more than one different sets of culture conditions.
  • a pool may be subdivided further into groups, either randomly or non- randomly; such groups are not themselves “pools" for the present purposes, but may themselves be pooled by combination, for example after exposure to different sets of culture conditions.
  • Proliferation Cell growth and cell proliferation are used interchangeably herein to denote multiplication of cell numbers without differentiation into different cell types or lineages. In other words, the terms denote increase of viable cell numbers.
  • proliferation is not accompanied by appreciable changes in phenotype or genotype.
  • Differentiation Cell differentiation is the development, from a cell type, of a different cell type.
  • a bipotent, pluripotent or totipotent cell may differentiate into a neural cell. Differentiation may be accompanied by proliferation, or may be independent thereof.
  • the term 'differentiation' generally refers to the acquisition of a phenotype of a mature cell type from a less developmentally defined cell type, e.g. a neuron, or a lymphocyte, but does not preclude transdifferentiation, whereby one mature cell type may convert to another mature cell type e.g. a neuron to a lymphocyte.
  • the differentiation state of a cell is the level to which a cell has differentiated along a particular pathway or lineage.
  • the state of a cellular process refers to whether a cellular process is occurring or not and in complex cellular processes can denote a particular step or stage in that cellular process.
  • a cellular differentiation pathway in a cell may be inactive or may have been induced and may comprise a number of discrete steps or components such as signalling events characterised by the presence of a characteristic set of enzymes or intermediates.
  • a gene is a nucleic acid which encodes a gene product, be it a polypeptide or an RNA gene product.
  • a gene includes at least the coding sequence which encodes the gene product; it may, optionally, include one or more regulatory regions necessary for the transcription and/or translation of the coding sequence.
  • Gene Product A gene product is typically a protein encoded by a gene in the conventional manner. However, the term also encompasses non-polypeptide gene products, such as ribonucleic acids, which are encoded by the gene.
  • Nucleic acid synthesis Nucleic acids may be synthesised according to any available technique. Preferably, nucleic acid synthesis is automated. Moreover, nucleic acids may be produced by biological replication, such as by cloning and replication in bacterial or eukaryotic cells, according to procedures known in the art.
  • Differential Expression Genes which are expressed at different levels in response to cell culture conditions can be identified by gene expression analysis, such as on a gene array, by sequencing such as RNA-seq, or by any methods known in the art. Genes which are differentially expressed display a greater or lesser quantity of mRNA or gene product in the cell under the test conditions than under alternative conditions, relative to overall gene expression levels.
  • Transfection Genes may be transfected into cells by any appropriate means.
  • the term is used herein to signify conventional transfection, for example using calcium phosphate, but also to include other techniques for transferring nucleic acids into a cell, including transformation, viral transduction, electroporation and the like.
  • modulation is used to signify an increase and/or decrease in the parameter being modulated.
  • modulation of gene expression includes both increasing gene expression and decreasing gene expression.
  • Stem Cells Stem cells are described in detail in Stem Cells: Scientific Progress and Future Research Directions. Department of Health and Human Services. June 2001. http://www.nih.gov/news/stemcell/scireport.htm. The contents of the report are herein incorporated by reference.
  • Stem cells are cells that are capable of differentiating to form at least one and sometimes many specialised or differentiated cell types.
  • the repertoire of the different cells that can be formed from stem cells is thought to be exhaustive; that is to say it includes all the different cell types that make up the organism.
  • Stem cells are present throughout the lifetime of an organism, from the early embryo where they are relatively abundant, to the adult where they are relatively rare.
  • Stem cells present in many tissues of adult animals are important in normal tissue repair and homeostasis. The existence of these cells has raised the possibility that they could provide a means of generating specialised functional cells that can be transplanted into humans and replace dead or non-functioning cells in diseased tissues.
  • the list of diseases for which this may provide therapies includes Parkinson's disease, diabetes, spinal cord injury, stroke, chronic heart disease, end-stage kidney disease, liver failure and cancer.
  • spermatogonial stem cells are unipotent as they naturally produce only spermatozoa, whereas haematopoietic stem cells are multipotent, and embryonic stem cells are thought to be able to give rise to all cell types and are said to be totipotent or pluripotent.
  • embryonic stem cells are thought to be able to give rise to all cell types and are said to be totipotent or pluripotent.
  • three types of mammalian pluripotent stem cell have been isolated. These cells can give rise to cell types that are normally derived from all three germ layers of the embryo (endoderm, mesoderm and ectoderm).
  • the three types of stem cell are: embryonal carcinoma (EC) cells, derived from testicular tumours; embryonic stem (ES) cells, derived from the pre-implantation embryo (normally the blastocyst); and embryonic germ (EG) cells derived from the post-implantation embryo (normally cells of the foetus destined to become part of the gonads).
  • EC embryonal carcinoma
  • ES embryonic stem
  • EG embryonic germ
  • transdifferentiation is the conversion of one differentiated cell type to another, with or without an intervening cell division. Differentiation can sometimes be reversed or altered.
  • in vitro protocols are now available in which cell lines can be induced to transdifferentiate.
  • specialised cell types can de- differentiate to yield stem-like cells with the potential to differentiate into further cell types.
  • derived insulin producing cells from human pancreatic ductal cells by: 1) selecting ductal cells over islet cells by selective adhesion on a solid surface in the presence of serum for 2-4 days; 2) subsequently withdrawing serum and adding keratinocyte growth factor to select for ductal epithelial cells over fibroblasts for 5-10 days; and 3) overlaying the cells with the extracellular matrix preparation 'Matrigel' for 3- 6 weeks.
  • serial cell culture Lumelsky et al.
  • derived insulin secreting cells by directed differentiation of mouse embryonic stem (ES) cells by: 1) expanding ES cells in the presence of LIF for 2-3 days; 2) generating embryoid bodies in the absence of LIF over 4 days; 3) selecting nestin-positive cells using ITSFn medium for 6-7 days; 4) expanding pancreatic endocrine precursors in N2 medium containing 827 media supplement and bFGF for 6 days; and 5) inducing differentiation to insulin secreting cells by withdrawing bFGF and adding nicotinamide.
  • ES mouse embryonic stem
  • stem cells Many of the factors that have been found to influence self-renewal and differentiation of stem cells in vitro are naturally-occurring molecules. This is to be expected, as differentiation is induced and controlled by signalling molecules and receptors that act along signal transduction pathways. However, by the same token, it is likely that many synthetic compounds will have an effect on stem cell differentiation. Such synthetic compounds that have high probability of interacting with cellular targets within signalling and signal transduction pathways (so called drugable targets) are routinely synthesised, for instance for drug screening by pharmaceutical companies. Once known, these compounds can be used to direct the differentiation of stem cells ex vivo, or can be administered in vivo in which case they would act on resident stem cells in the target organ of a patient.
  • tissue culture In developing conditions for the successful culture of a particular cell type, or in order to achieve or modulate a cellular process, it is often important to consider a variety of factors. One important factor is the decision of whether to propagate the cells in suspension or as a monolayer attached to a substrate. Most cells prefer to adhere to a substrate although some, including transformed cells, haematopoietic cells, and cells from ascites, can be propagated in suspension.
  • adhesion substrate Assuming the culture is of adherent cells, an important factor is the choice of adhesion substrate.
  • Most laboratories use disposable plastics as substrates for tissue culture.
  • the plastics that have been used include polystyrene (the most common type), polyethylene, polycarbonate, Perspex, PVC, Teflon, cellophane and cellulose acetate. It is likely that any plastic can be used, but many of these will need to be treated to make them wettable and suitable for cell attachment. Furthermore it is very likely that any suitably prepared solid substrate can be used to provide a support for cells, and the substrates that have been used to date include glass (e.g.
  • alum-borosilicate and soda-lime glasses alum-borosilicate and soda-lime glasses
  • rubber synthetic fibres
  • polymerised dextrans metal (e.g. stainless steel and titanium) and others.
  • cell types such as bronchial epithelium, vascular endothelium, skeletal muscle and neurons require the growth substrate to be coated with biological products, usually extracellular matrix materials such as fibronectin, collagen, laminin, polylysine or others.
  • the growth substrate and the method of application can have an effect on cellular processes such as the growth and differentiation characteristics of cells, and these must be determined empirically as discussed above. Probably the most obviously important of the variables in cell culture is the choice of culture medium and supplements such as serum.
  • aqueous compartment for cell growth, complete with nutrients and various factors, some of which have been listed above, others of which are poorly defined. Some of these factors are essential for adhesion, others for conveying information (e.g. hormones, mitogens, cytokines) and others as detoxificants.
  • Commonly used media include RPMI 1640, MEM/Hank's salts, MEM/Earle's salts, F12, DMEM/F12, L15, MCDB 153, and others.
  • the various media can differ widely in their constituents - some of the common differences include sodium bicarbonate concentration, concentration of divalent ions such as Ca and Mg, buffer composition, antibiotics, trace elements, nucleosides, polypeptides, synthetic compounds, drugs, etc.
  • the gas phase of the tissue culture is also important and its composition and volume which should be used can depend on the type of medium used, the amount of buffering required, whether the culture vessel is open or sealed, and whether a particular cellular process needs to be modulated. Common variables include concentration of carbon dioxide and oxygen.
  • Other conditions important to tissue culture include the choice of culture vessel, amount of headspace, inoculation density, temperature, frequency of media changes, treatment with enzymes, rate and mode of agitation or stirring. Varying the cell culture conditions is therefore a method of achieving a desired cellular process.
  • One aspect of the invention recognises that variation of the cell culture conditions in a serial manner can be a highly effective method for achieving a cellular effect.
  • tissue culture conditions are required to effect a cellular process.
  • the different conditions may include additions or withdrawals to/from the media or the change of media at specific time points.
  • Such a set of conditions examples of which are given below, are commonly developed by trial end error as has been discussed above.
  • test units An aspect of the present invention is that, in some embodiments, groups of cells (cell colonies) can be grown in cell culture under various conditions and that the colony can largely maintain its integrity under various conditions, when disturbed, and when mixed with other colonies. Such groups or colonies are referred to herein as test units. In some instances, test units are a single cell, in which case it is not necessary to form cell colonies.
  • test units may be achieved, by way of illustration, by growing cells as adherent cultures on solid substrates such as carriers. If cell proliferation occurs after seeding on the carriers, the daughter cells will attach on the same carrier and form part of the same colony. In general, live adherent cells do not readily dissociate from their growth substrate, and so the integrity of the cell colony persists despite any mechanical manipulation of the carrier, agitation of the culture medium, or transfer into another tissue culture system.
  • tissue culture can be miniaturised: relatively few cells are required to colonise a microcarrier bead (see below) compared to even the smallest tissue culture flask.
  • a further advantage of growing cell units formed on carriers is that cell culture can be scaled up. Growth of stem cells on carriers offers a way of scaling up production to provide enough material for stem cell therapy. Equally, differentiation of stem cells on carriers offers a way of scaling up production as differentiation proceeds, eventually providing enough material for cell replacement therapies. Scale up of such cell cultures requires at least 50g (dry weight) of microcarrier, preferably, 100g, 500g, 1 kg, 10kg or more.
  • Another important advantage of forming cell units on solid substrates is that the substrate - and therefore the attached cells by reason of association - can be labelled by various means.
  • Glass spheres of 3mm and 5mm have been widely used as cell adhesion substrates, particularly in glass bead bioreactors (e.g. such as manufactured by Meredos Gmbh) used for the scale-up of cell cultures. These beads are typically used in packed beds rather than batch culture, to avoid mechanical damage to the adherent cells. In contrast, when cells are grown on smaller carriers they can be treated as a suspension culture.
  • microcarrier cell culture A common method of growing cells on small carriers is referred to as microcarrier cell culture (see 'Microcarrier cell culture, Principles and Methods', Edition AA, available from Amersham Biosciences (18-1140-62); herein incorporated in its entirety by reference).
  • Microcarrier cultures are used commercially for antibody and interferon production in fermenters of up to 4000 litres.
  • a variety of microcarriers is available, ranging in shape and size and made of different materials.
  • Microcarrier beads made of polystyrene (Biosilon, Nunc), glass (Bioglass, Solohill Eng), collagen(Biospheres, Solohill Eng), DEAE sephadex (Cytodex-1 , Pharmacia), dextran (Dormacell, Pfeifer & Langen), cellulose (DE-53, Whatman), gelatin (Gelibead, Hazelton Lab), and DEAE dextran (Microdex, Dextran Prod.) amongst others are commercially available. These carriers are well characterised in terms of the specific gravity of the beads, the diameter and the surface area available for cell growth. In addition a number of porous (micro) carriers are available with greatly increased surface area for cell growth.
  • porous carriers are suitable for growth of both anchorage dependent cells, as well as for suspension cells which are carried by entrapment in the network of open, interconnecting pores.
  • Porous carriers are available in materials such as gelatin (Cultispher G, HyClone), cellulose (Cytocell, Pharmacia), polyethylyne (Cytoline 1 and 2, Pharmacia), silicone rubber (Immobasil, Ashby Scientific), collagen (Microsphere, Cellex Biosciences), and glass (Siran, Schott Glassware). These carriers are variously suited to stirred, fluidised or fixed bed culture systems.
  • cells may be grouped by immurement, i.e. confined within a medium permeable barrier.
  • Membrane culture systems have been developed where a permeable dialysis membrane retains a group of cells, but allows the culture medium and its constituents to exchange freely with the inner and outer compartments.
  • Cell culture in hollow fibre cartridges has also been developed, and a multitude of fibres and even turn-key systems are commercially available (e.g. from Amicon, Cellex Biosciences).
  • Cell encapsulation in semi-solid matrices has also been developed, where cells are immobilised by adsorption, covalent bonding, crosslinking or entrapment in a polymeric matrix.
  • Materials that have been used include gelatin, polylysine, alginate and agarose.
  • a typical protocol is to mix 5% agarose at 40°C with a suspension of cells in their normal growth medium, to emulsify the mixture using an equal volume of paraffin oil and to cool in an ice bath, producing spheres of 80-200pm diameter. These spheres can be separated from the oil and transferred to medium in a tissue culture vessel.
  • Cell entrapment is a simple method for the immobilisation of groups of cells, akin to the use of microcarriers or porous substrates.
  • a simple technique is to enmesh cells in cellulose fibres such as DEAE, TLC, QAE, TEAE (all available from Sigma).
  • Other more sophisticated devices are ceramic cartridges which are suitable for suspension cells, as in the Opticel culture system (Cellex Biosciences).
  • Test units can be associated with a particular factor including, but without limitation, proteins, nucleic acids or other chemicals such as drugs. Pre-conditioning of substrates can be achieved in many ways, for instance simply by incubating the substrate with the factor of interest, or by attaching the factor covalently or non-covalently to the substrate. Soluble factors can be incorporated into dry materials by impregnation. This technique relies on the rapid ingress of liquid, carrying soluble factors, into dry porous material that concomitantly becomes swollen and ready for use. Solid factors can be incorporated for example by mixing the factor in fibrinogen with thrombin solution, at which point a fibrin clot containing the factor is formed. Multiple other ways can be envisaged of associating factor(s) with a cell group, in addition to impregnating, entrapping or encapsulating the factor together with cells.
  • a method for associating a cell group with a number of different factors is to pre-form cocktails of factors which are subsequently associated with a particular cell group.
  • a second method would be to serially condition cell groups in a number of factors. Using dry formulations of cell group growth substrates as an example, this method would involve firstly partially swelling the substrate in a solution containing a first factor and subsequently further swelling the same in a solution containing a second factor, resulting in a substrate to which both factors have become associated.
  • Factors leaking into the growth medium are diluted to such an extent that their concentration falls below physiologically relevant limits and they have no effect on any additional cell group to which they are exposed.
  • the diffusion of the factor out of e.g. the substrate forming part of the cell unit is governed by parameters such as the nature and dimensions of the material, the mean pore diameter, and the molecular weight and concentration of the factor.
  • factor release can be measured by physical assays such as HPLC analysis or release of labelled factor into the medium, or by biological assays such as the dorsal root ganglion outgrowth bioassay for neurotrophic factors.
  • Combinatorial serial culture of cells Split-pool cell culture Forming test units is furthermore useful for sampling multiple tissue culture conditions as each cell unit constitutes an easily handled unit that can be exposed to a variety of cell culture conditions.
  • cell groupings are produced by growing cells in microcarrier culture, and the terms test unit, cell unit, cell group, single cell, colony and bead are used interchangeably.
  • test unit, cell unit, cell group, single cell, colony and bead are used interchangeably.
  • the methods described are equally applicable to any test unit, for instance those described above.
  • a particularly efficient method for sampling a large number of cell culture conditions is referred to as Combinatorial Cell Culture or split-pool cell culture (Fig 1) and in one embodiment involves the serial subdividing and combining of groups of cell units in order to sample multiple combinations of cell culture conditions.
  • the method operates by taking an initial starter culture (or different starter cultures) of cell units divided into X number of aliquots each containing multiple beads (groups/colonies/carriers/cells) which are grown separately under different culture conditions. Following cell culture for a given time, the cell units can be pooled by combining and mixing the beads from the different aliquots.
  • This pool can be split again into X2 number of aliquots, each of which is cultured under different conditions for a period of time, and subsequently also pooled.
  • This iterative procedure of splitting, culturing and pooling (or pooling, splitting and culturing; depending on where one enters the cycle) cell units allows systematic sampling of many different combinations of cell culture conditions.
  • the complexity of the experiment is equal to the product of the number of different conditions (X1 x X2 x ...Xn) sampled at each round. Note that the step of pooling all the cell units prior to a subsequent split can be optional - a step in which a limited number of cell units are pooled can have the same effect.
  • the invention therefore embodies a number of related methods of systematically sampling multiple combinations of cell culture conditions where groups of test units are handled in bulk. Regardless of the precise manner in which a diversity of cell culture conditions is sampled by this means the procedure is efficient because multiple cell units can share a single vessel, where they are cultured under identical conditions, and it can be carried out using only a few culture vessels at any one time (the number of culture vessels in use is equal to the number of split samples).
  • This procedure can be used to sample growth or differentiation conditions for any cell type, or the efficiency of biomolecule production (e.g. production of erythropoeitin or interferon) by any cell type. Because the procedure is iterative, it is ideally suited to testing multistep tissue culture protocols - for instance those described above in connection with stem cell differentiation.
  • the variables which can be sampled using this technique include cell type, cell grouping (e.g. microcarrier culture, cell encapsulation, whole organism), growth substrate (e.g. fibronectin on microcarrier), duration of cell culture round, temperature, different culture media (including different concentrations of constituents), growth factors, conditioned media, co-culture with various cell types (e.g.
  • RNAi RNAi, triple helix
  • sensory inputs in the case of organisms
  • split-split cell culture The purpose of performing split-pool processes on cell units is to systematically expose these to a pre-defined combination of conditions. The person skilled in the art will conceive of many different means of achieving this outcome. In addition to splitpool processes and variations thereof, it is worthwhile briefly discussing split-split processes.
  • a split-split process involves subdividing a group of cell units at least twice, without intervening pooling of cell units. If split-split processes are used over a large number of rounds, the number of separate samples that are generated increases exponentially. In this case it is important to employ some level of automation, for example the use of a robotic platform and sophisticated sample tracking systems.
  • split-split steps are that (since cell units are not combined) it is possible to segregate lineages of the various cell units based on their cell culture history. Consequently split-split steps can be used to deduce if a particular cell culture condition is responsible for any given cellular process and therefore used to deduce the culture history of cell units.
  • the splitting and/or pooling of cell units may be accomplished totally randomly or may follow a predetermined protocol. Where cell units are split and/or pooled randomly, the segregation of a given cell unit into any group is not predetermined or prejudiced in any way. In order to result in a high probability that at least one cell unit has been exposed to each of the possible combinations of cell culture conditions, it is advantageous to employ a larger number of cell units than the total number of combinations of cell culture conditions that are being tested. Under certain circumstances it is therefore advantageous to split and/or pool cell units according to a predetermined protocol, the overall effect being that adventitious duplications or omissions of combinations are prevented.
  • Predetermined handling of cell units can be optionally planned in advance and logged on a spreadsheet or computer programme, and splitting and/or pooling operations executed using automated protocols, for instance robotics.
  • Labelling of cell units can be by any of a number of means, for instance labelling by RFID, optical tagging or spatial encoding.
  • Robotic devices capable of determining the identity of a sample, and therefore partitioning the samples according to a predetermined protocol, have been described (see 'Combinatorial Chemistry, A practical Approach', Oxford University Press (2000), Ed H. Fenniri).
  • standard laboratory liquid handling and/or tissue culture robotics (for example such as manufactured by: Beckman Coulter Inc, Fullerton, CA; The Automation Partnership, Royston, UK) is capable of spatially encoding the identity of multiple samples and of adding, removing or translocating these according to pre-programmed protocols.
  • the cell units can be studied to observe any given cellular process that may have been affected by the tissue culture conditions.
  • the examples below are illustrative and not intended to limit the scope of the invention.
  • the cell units can be assayed to determine whether there are members displaying increased cell proliferation. This can be achieved by a variety of techniques, for instance by visual inspection of the cell units under a microscope, or by quantitating a marker product characteristic of the cell. This may be an endogenous marker such as a particular DNA sequence, or a cell protein which can be detected by a ligand or antibody. Alternatively an exogenous marker, such as green fluorescent protein (GFP), can be introduced into the cell units being assayed to provide a specific readout of (living) cells.
  • GFP green fluorescent protein
  • Live cells can be visualised using a variety of vital stains, or conversely dead cells can be labelled using a variety of methods, for instance using propidium iodide.
  • the labelled cell units can be separated from unlabelled ones by a variety of techniques, both manual and automated, including affinity purification ('panning'), or by fluorescence activated cell sorting (FACS) or broadly similar techniques (Fig. 17).
  • affinity purification 'panning'
  • FACS fluorescence activated cell sorting
  • Fig. 17 broadly similar techniques
  • certain analysis and sorting instruments e.g. see Union Biometrica Inc., Somerville MA, USA
  • the cell units can be assayed to determine whether there are members displaying a particular genotype or phenotype. Genotype determination can be carried out using well known techniques such as the polymerase chain reaction (PCR), fluorescence in situ hybridisation (FISH), DNA sequencing, and others. Phenotype determination can be carried out by a variety of techniques, for instance by visual inspection of the cell units under a microscope, or by detecting a marker product characteristic of the cell.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridisation
  • Phenotype determination can be carried out by a variety of techniques, for instance by visual inspection of the cell units under a microscope, or by detecting a marker product characteristic of the cell.
  • This may be an endogenous marker such as a particular DNA or RNA sequence, or a cell protein which can be detected by a ligand, conversion of an enzyme substrate, or antibody that recognises a particular phenotypic marker (For instance see Appendix E of Stem Cells: Scientific Progress and Future Research Directions. Department of Health and Human Services. June 2001 ; appendices incorporated herein by reference).
  • a genetic marker may also be exogenous, i.e. one that has been introduced into the cell population, for example by transfection or viral transduction. Examples of exogenous markers are the fluorescent proteins (e.g. GFP) or cell surface antigens which are not normally expressed in a particular cell lineage or which are epitope-modified, or from a different species.
  • a transgene or exogenous marker gene with associated transcriptional control elements can be expressed in a manner that reflects a pattern representative of an endogenous gene(s). This can be achieved by associating the gene with a minimal cell type-specific promoter, or by integrating the transgene into a particular locus (e.g. see European patent No. EP 0695351).
  • the labelled cell units can be separated from unlabelled ones by a variety of techniques, both manual and automated, including affinity purification ('panning'), or by fluorescence activated cell sorting (FACS).
  • affinity purification 'panning'
  • FACS fluorescence activated cell sorting
  • An alternative or complementary technique for enriching cell units of a particular genotype or phenotype is to genetically select the desired groups. This can be achieved for instance by introducing a selectable marker into the cell units, and to assay for viability under selective conditions, for instance see Soria et al (2000, Diabetes vol 49, p1-6) who used such a system to select insulin secreting cells from differentiated ES cells. Li et al (1998, Curr Biol vol 8, p 971-974) identified neural progenitors by integrating the bifunctional selection marker/reporter f3geo (which provides for f3- galactosidase activity and G418 resistance) into the Sox2 locus by homologous recombination in murine ES cells.
  • neural progenitors Since one of the characteristics of neural progenitors is expression of Sox2, and therefore the integrated marker genes, these cells could be selected from non-neuronal lineages by addition of G418 after inducing differentiation using retinoic acid. Cell viability could be determined by inspection under a microscope, or by monitoring f3-gal activity. Unlike phenotype-based selection approaches, which can be limited by the availability of an appropriate ligand or antibody, genetic selection can be applied to any differentially expressed gene.
  • Determination of the identity or cell culture history of a cell unit When handling large numbers of cell units, their identity and/or cell culture history (for example the chronology and the exact nature of a series of culture conditions that any one group or unit may have been exposed to) can become confused. For instance, the split-pool protocol of cell culture necessarily involves mixing cell units in each round, making it difficult to follow individual units. Determining the cell culture history of a cell unit in a mixture of cell units which have been subjected to multiple culture conditions is sometimes referred to as 'deconvolution' of the cell culture history. One method of doing this is to label cell units and it is therefore advantageous to label the cell units.
  • Labelling may be performed at the beginning of an experiment, or during each round of an experiment and may involve a unique label (which may or may not be modified in the course of an experiment) or a series of labels which comprise a unique aggregate. Similarly, reading of the label(s) may take place during each round or simply at the end of the experiment. Preferably, unique labels such as RFID labels are read during each round, whereas labels added serially at each round are read at the end of an experiment. Labelling of cell units may be achieved by a variety of means, for instance labelling either the cells themselves, or any material to which the cells are attached or otherwise associated with.
  • One method of labelling cell units involves associating cell units with a tag that becomes sequentially modified as it is placed in different culture conditions. This may involve for instance- the addition or subtraction of further units to the tag such that its stereochemistry, sequence or mass is altered; or the alteration of electronic memory as in read-write RF transponders (see below).
  • Another method of labelling cell units involves sequentially associating unique tags with the cell units whenever they are cultured under different conditions, such that subsequent detection and identification of the tags provides for an unambiguous record of the chronology and identity of the cell culture conditions to which the cell unit has been exposed.
  • Tags can be taken up by cells, or attached to the cell surface by adsorption, or a suitable ligand or antibody, or conjugated to a cell-associated matrix such as a carrier by adsorption, colloidal forces or a variety of linkages such as covalent linkage or non- covalent linkage, e.g. biotin-streptavidin linkage.
  • a cell-associated matrix such as a carrier by adsorption
  • colloidal forces or a variety of linkages such as covalent linkage or non- covalent linkage, e.g. biotin-streptavidin linkage.
  • one simple tag that can be introduced to cells or attached to a matrix associated with cells is an oligonucleotide of defined length and/or sequence.
  • Oligonucleotides may comprise any class of nucleic acid (e.g. RNA, DNA, PNA, linear, circular or viral) and may contain specific sequences for amplification (e.g. primer sequences for PCR) or labels for detection (e.g. fluorophores or quenchers, or isotopic tags).
  • the detection of these may be direct, for instance by sequencing the oligos or by hybridising them to complementary sequences (e.g. on an array or chip), or indirect as by monitoring an oligonucleotide-encoded gene product, or the interference of the nucleotide with a cellular activity (e.g. antisense inhibition of a particular gene).
  • nucleic acid tags can comprise RCA templates, elongation primers, or struts that aid the circularization of minicircle templates).
  • Any molecular or macromolecular tag can be used so long as it can be detected, including peptide tags, coloured or fluorescent compounds, secondary amines, halocarbons, mixtures of stable isotopes etc.
  • Tags may attach to cell units directly or via an intermediary, for instance an antibody raised against a component of the cell unit, or via an interacting pair such as biotin-steptavidin.
  • tags can be protected against degradation by the components of the cell culture, for example by chemical or other modification or by encapsulation.
  • Encapsulation of tags can take place in many different media, for example in beads many types of which are available from suppliers such as Bangs Laboratories Inc. (Fishers IN, USA), and encapsulation may be used to standardise tag dosage in addition to providing components for tag amplification and/or detection (for example by providing PCR primers for use with a DNA tag).
  • a preferred method of labelling cell units employs fluorescent beads such as those manufactured by Luminex Corporation (Austin, TX, USA).
  • the Luminex system comprises polystyrene beads which may or may not be externally derivatised (e.g.
  • a further preferred method employs beads such as those manufactured by Bangs Laboratories Inc. (Fishers IN, USA).
  • the Bangs system comprises bead sets which can be distinguished based on differing sizes (e.g. bead sets of 4.4pm and 5.5pm diameter). Beads within each set can be furthermore distinguished from each other based on differing fluorescence intensity owing to differential loading with a single fluorescent dye. It is possible to use many different dyes with different absorption or emission characteristics, which can be internally loaded or attached externally to carriers by a multiplicity of means. It is furthermore possible to use 'quantum dots' to obtain a very high number of different fluorescent labels which can be read conveniently.
  • a preferred labelling technique comprises nucleic acid modification of the cell by insertion of a nucleic acid “barcode”, by viral infection (such as by a lentivirus) or gene editing, for instance by CRISPR.
  • Cell growth substrates such as those described in connection with forming cell units can be derivatised or coated with substances that facilitate tagging and do not interfere with cell growth.
  • a preferred method of derivatising carriers is to modify them covalently or non- covalently with biotin, to which a tag can be attached via streptavidin or avidin.
  • biotin to which a tag can be attached via streptavidin or avidin.
  • an inert tag i.e. an inert tag
  • More complicated molecular tagging strategies can also be envisaged, including the strategy of 'binary encoding' where information is recorded by a set
  • Detection of tags can be accomplished by a variety of methods familiar to those skilled in the art. Methods include mass spectrometry, nuclear magnetic resonance, sequencing, hybridisation, antigen detection, electrophoresis, spectroscopy, microscopy, image analysis, fluorescence detection, etc. Of particular interest are labelling or encoding strategies in which labelling is carried out only once or where labelling and/or detection are non-physical and therefore non- invasive. Radiofrequency Identification (RFID) is an example of a system exhibiting these properties. RFID employs transponders (RF tags), antennae and readers.
  • RFID Radiofrequency Identification
  • An RF tag is a small electronic circuit, usually encased in glass or plastic, which in its simplest form provides access to a unique identification code that may be 'read', without contact or line of sight, by suitable electronics. Tags may also store information generated by the user, again without contact or line of sight.
  • a 'reader' is an electronic unit that transfers information to and from one or more tags (it should be noted that the term reader is used interchangeably to mean both a read only and read/write unit). The size and features of a reader may vary considerably, and it may operate in isolation, or be connected to a remote computer system.
  • An antenna is used to transmit information from a reader to a tag, and to receive information sent by an RF tag.
  • An RFID system may operate in isolation, or be connected to a remote computer for more comprehensive interpretation and manipulation of identification and associated data derived from a tag.
  • One RFID strategy used in combinatorial chemistry is described in Nicolaou et al (1995, Angew Chem Inti Ed Engl, vol. 34, p. 2289) and comprises: (i) a porous enclosure containing a synthesis substrate and the semiconductor tag; (ii) the solid phase synthesis resin; (iii) a glass- encased Single or Multiple Addressable Radiofrequency Tag semiconductor unit capable of receiving, storing and emitting radiofrequency signals.
  • a similar device could be adapted to growing and following cell units simply by replacing the solid phase synthesis resin with tissue culture microcarriers or suitable cell units. More variations of this can be envisaged including but not limited to (coated or uncoated) RF tags on which cells are grown directly, or RF tags implanted into cell units or organisms.
  • tags do not necessarily have to be distinguished by their chemical or molecular structure in the first instance.
  • Multiple variations of the non-chemical tagging strategy can be devised to determine the identity of a given cell unit in a mixture or of deducing the identity of the different cell units that comprise a mixture.
  • optical or visual methods of tagging have been described where different shaped objects, graphically encoded objects or different colours denote the identity of a sample (for example see1998, Guiles et al, Angew. Chem. Inti Ed Engl, vol.
  • a further method of tracking or labelling cell units is to encode their identity spatially, i.e. by their position in space. In this method different cell units are segregated in defined relative positions, and these positions denote or encode the identity of the units.
  • cell units may be cultured in an array, whereby the identity and/or culture history of each unit is known and is associated to a particular position in the array.
  • arrays can comprise collections of tissue culture flasks, wells of a multi-well plate, or locations on a glass slide or other surface. Examples of positional encoding strategies can be found in Geysen et al. (1984, Proc Natl Acad Sci USA vol. 81 , p. 3998-4002), Fodor et al. (1991 , Science vol. 251 , p. 767-773), Ziauddin and Sabatini (2001 , Nature, Vol. 411 , p. 107-110), and Wu et al. (2002, Trends Cell Biol. Vol. 12(10), p.485-8).
  • the invention has many facets, each of which may have many forms that may be combined to form numerous permutations of the invention.
  • cell units are preferably labelled. Labelling of a cell unit allows the derivation of useful information from the experiment regarding the outcome of the particular conditions sampled by the labelled cell unit, as opposed to all the cell units.
  • Cell reprogramming involves changing the identity or function of a cell. This can involve transforming a cell from one type to another or reverting mature cells back to a more pluripotent or stem cell-like state, before re-differentiating into a more committed state.
  • reprogramming is the conversion of a differentiated, committed cell type into another differentiated, committed cell type. Reprogramming excludes mere conversion of a pluripotent cell into a differentiated cell type.
  • reprogramming as referred to herein may encompass any one or more of these techniques.
  • iPSCs Induced Pluripotent Stem Cells
  • pluripotent cells induce the mature cells to revert to a pluripotent state, similar to embryonic stem cells.
  • the pluripotent cells can then be further differentiated into a desired cell type.
  • Yamanaka used retroviral vectors to deliver TFs but many other methods are available, including non-integrating viral vectors, mRNA delivery, plasmids and direct addition of TF proteins, as well as the use of non-coding RNA.
  • ncRNAs Various types of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (IncRNAs), and others, can be involved in regulating transcription factors. These ncRNAs can be used to replace TFs in the assays of then invention.
  • microRNAs miRNAs
  • miRNAs are short ( ⁇ 22 nucleotides) ncRNAs that generally bind to the 3' untranslated region (UTR) of target mRNAs, leading to their degradation or translation repression. Through this mechanism, miRNAs can negatively regulate the levels of specific transcription factors (Bartel DP. (2009) MicroRNAs: target recognition and regulatory functions. ‘Cell*. 136(2):215-33).
  • IncRNAs Long non-coding RNAs
  • Some IncRNAs can interact with transcription factors directly, modulating their activity, and can thus serve to modulate TF activity in an assay according to the invention. See Rinn JL, Chang HY. (2012) Genome regulation by long noncoding RNAs. Annual Review of Biochemistry. 81 :145-66.
  • Non-coding RNAs have been shown to play roles in conjunction or as an alternative to the classic Yamanaka factors:
  • miR-302/367 Cluster This cluster of microRNAs has been shown to enhance reprogramming efficiency when used in combination with the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc). See Anokye-Danso F, et al. (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 8(4):376- 88.
  • fibroblasts can be directly converted to neurons, cardiomyocytes, or other cell types, depending on the factors used. Many techniques are currently available for transdifferentiation:
  • lineage-specific transcription factors are delivered to cells. For example:
  • RNA-based methods can alleviate concerns associated with potential DNA integrations into the host genome. Warren, et al., (2010) Cell Stem Cell, 7(5), 618-630. 3. Small Molecules and Chemicals:
  • the CRISPR/Cas9 system originally known for its gene-editing capabilities, can be adapted for gene activation by using a deactivated Cas9 (dCas9) fused to transcriptional activators. This can be used to activate endogenous lineage-specific genes, promoting transdifferentiation.
  • dCas9 deactivated Cas9
  • MicroRNAs (miRNAs):
  • Direct lineage conversion involves significant changes in the epigenetic landscape of a cell. Incorporating molecules that modulate epigenetic marks can increase the efficiency of transdifferentiation. Polo, Jet al., (2012) Cell, 151 (7), 1617-1632
  • Extracellular Cues and Microenvironment While transcription factor-mediated conversion is the primary driver, the efficiency and success of the conversion process can be influenced by the microenvironment, including extracellular matrix components, neighboring cells, and culture conditions. Engler, A. J., et al., (2006). Cell, 126(4), 677-689.
  • reprogramming is preferably transdifferentiatoin, by which cells are converted directly from one differentiated state to another, without passing through a pluripotent state.
  • a “cell type”, as referred to herein, is a differentiated cell belonging to a specified lineage.
  • a cell type is not a totipotent cell.
  • a cell type is not a pluripotent cell capable of differentiating into more than one lineage.
  • a cell lineage may be any of a variety of cells types which can develop during normal embryogenesis. Examples include:
  • Hematopoietic Lineage This lineage gives rise to all the different blood cells.
  • Hematopoietic stem cells (HSCs) in the bone marrow differentiate into various types of blood cells, such as erythrocytes (red blood cells), various leukocytes (neutrophils, lymphocytes, and monocytes), and platelets.
  • Neuronal Lineage Neural stem cells differentiate into various types of neuronal cells and glial cells. This includes motor neurons, sensory neurons, astrocytes, oligodendrocytes, and microglia.
  • Epidermal Lineage The epidermal stem cells in the skin differentiate into various skin cells, including keratinocytes, melanocytes, and cells of the hair follicles.
  • Myogenic stem cells can differentiate into muscle fibres or myocytes.
  • MSC Mesenchymal Stem Cell Lineage: MSCs are multipotent and can give rise to a variety of cell types, including osteoblasts, chondrocytes, fibrobalsts and adipocytes.
  • Intestinal Lineage Stem cells in the crypts of the intestines differentiate into various types of intestinal cells, such as absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells.
  • Liver stem cells can differentiate into hepatocytes, which are the main functional cells of the liver, and cholangiocytes, which line the bile ducts.
  • Cardiac Lineage Cardiac progenitor cells can differentiate into various cell types of the heart, such as cardiomyocytes, endothelial cells, and smooth muscle cells.
  • Germ Cell Lineage This lineage leads to the formation of ova in females and sperm in males.
  • Retinal progenitor cells can differentiate into various types of retinal cells, such as photoreceptors, bipolar cells, and ganglion cells.
  • Reprogramming can involve the conversion of a cell from one lineage to another, or from one cell type within a lineage to another cell type within the same lineage, for example from a fibroblast to an osteoblast.
  • test unit as referred to herein is a cell or group of cells which can be handled as a unit. It is synonymous with cell unit.
  • a test unit can be a cell unit as referred to in W02004031369, comprising cells arranged on a microcarrier. In a preferred embodiment, however, a test unit is a single cell.
  • test units comprise a carrier which in turn comprises one or more cells
  • the carrier may be populated with cells in a stochastic manner.
  • the intended cell number is low, such as one cell or two cells per unit, the probability of cells populating the test units may be reduced such that some units comprise no cells.
  • not all test units comprise cells.
  • the test units are single cells and do not comprise any carrier; in such an embodiment, all test units comprise a cell.
  • Reagents as referred to herein may be any chemical, small molecule, transcription factor, nucleic acid, protein, compound or ion which can interact with a cell.
  • reagents are transcription factors, small molecule or protein compounds, or nucleic acids.
  • reagents are transcription factors (TFs) or agents which influence transcription factor activity, such as ncRNAs.
  • “Labelling” can include any technique which is capable of individually labelling a cell. For example, reference is made to the techniques described above.
  • cells are labelled by genetic modification, such as by the introduction of retroviral markers or modification using genome editing techniques, as described below.
  • Deconvolution of labels refers to the identification of labels on a cell, and optionally the order in which the labels were acquired by the cell, and thereby determining which reagents or combinations of reagents the cell has been exposed to. Since the cell is labelled whenever it is exposed to a given reagent, the reagent leaves a “mark” on the cell which can be used to map the cell’s history in exposure to reagents as well as the timing of such exposure.
  • Predicting the most effective transdifferentiation-inducing reagents requires a combination of experimental data, computational modelling, and optimization techniques.
  • An algorithm can be used to interrogate the results of exposure of cells to given reagents, and thereby predict which reagents can be employed to induce a desired transdifferentiation in a given cell type.
  • the results of the reagent exposure should be presented as a consistent metric to the algorithm; for example, the results can be in the form of gene expression data, cell morphology scores, specific gene activation events, and the like.
  • Output from the algorithm may be used to select initial reagents for the performance of the method of the invention.
  • the output data can be used to inform further instances of the performance of the method.
  • Machine learning can be employed to better predict input reagents for desired transdifferentiation outcomes.
  • a database may be provided of gene expression data related to reagent, and gene regulatory networks constructed further comprising the machinelearning model configured to output a gene regulatory graph.
  • Identification of cell types can be performed by any suitable means, including genetic, morphological, immunochemical or other features.
  • Gene expression analysis includes the analysis of one or more reporter genes, as well as analysis based on a wide variety of cellular genes including using arrays which analyse expression of the entire genome.
  • gene expression analysis focusses on one or more genes which are markers for the desired cell type.
  • the method of the invention can be sued to determine such genes by analysing gene expression changes in cells which are exposed to reagents which are shown to lead to desired transdifferentiation outcomes by analysis of alternative markers of cell differentiation, such as immunochemical markers or cell morphology.
  • Genes which influence cell transdifferentiation can in turn be purposefully modulated in order to induce transdifferentiation; for example, information about a gene being modulated in response to exposure to a reagent can be used to select a bene modulation technique, such as genome editing or the like, to further modulate the gene in place of administration of the reagent.
  • a bene modulation technique such as genome editing or the like
  • CombiCult® has been successfully deployed in several cases, allowing the discovery of for example novel protocols for expansion of hematopoietic stem cells, differentiation of macrophages, neutrophils, natural killer cells, megakaryocytes, smooth muscle cells, and oligodendrocyte progenitors.
  • the present invention provides:
  • Bespoke computational analyses to define the input of molecules to be tested, including a machine learning algorithm to prioritize molecular drivers of cell fate acquisition and prevent contradictory or opposing pathways to be tested together;
  • a second computational pipeline to analyse the results of the combinatorial screen by identifying viral barcodes and their representation in the acquisition of cell fates of interest.
  • the results of one CombiCult screen can be used to select input TFs for a further screen, or alternatively to determine optimal TFs for a desired transdifferentiation protocol.
  • scRNA-seq single-cell RNA-sequencing
  • scATAC-seq single cell chromatin accessibility data
  • an algorithm for selection of TFs can process the results of screens as follows:
  • effect_data this could be gene expression profiles, cell phenotype changes, or any measurable metric post-exposure
  • Desired cell type profile desired_profile
  • reagentjist For each cell in celljist: a. For each reagent in reagentjist: i. Get the effect of this reagent on this cell from effect_data. ii. Compute the similarity score between the effect and the desired_profile. (This could be a correlation score, frequency metric, or any relevant measure of similarity.) b. Rank the reagents for the cell based on the similarity scores. c. Store the top-ranked reagent for this cell.
  • Machine learning may be applied to the algorithm, to further refine the selection of reagents.
  • a machine learning algorithm may be applied in the following manner:
  • Training dataset Contains cell identities, timing and identity of reagent exposure, and observed effects post-exposure.
  • Desired cell type profile desired_profile
  • Optimal reagent(s) for transdifferentiation optimal_reagents
  • Data Preprocessing a. Normalize and standardize the data (e.g., z-score normalization for gene expression levels). b. Split the dataset into training and validation sets.
  • Feature Selection a. Select relevant features (e.g., specific gene expression levels, cell markers). b. If dataset is high-dimensional, consider dimensionality reduction methods like PCA.
  • Model Selection a. Choose a suitable ML model. Regression models like Random Forest or Gradient Boosting Machines (GBM) are examples. b. Train the model on the training set. c. Validate the model on the validation set.
  • GBM Gradient Boosting Machines
  • Hyperparameter Tuning a. Use techniques like grid search or random search to find the optimal hyperparameters for the chosen model. b. Retrain the model using the optimal hyperparameters
  • Prediction a. Use the trained model to predict the effect of each reagent on transdifferentiation towards the desired cell type profile. b. Rank reagents based on their predicted effectiveness.
  • Model Interpretation (Optional but recommended): a. Use techniques like SHAP (SHapley Additive exPlanations) or feature importance scores to interpret which features (e.g., specific timings, cell markers) are most influential in the predictions.
  • SHAP SHapley Additive exPlanations
  • feature importance scores to interpret which features (e.g., specific timings, cell markers) are most influential in the predictions.
  • the algorithm of the invention applies gold standard data pre-processing steps and machine learning methods to identify temporal (or pseudo-temporal) trajectories and gene expression changes associated with them. These trajectories will connect stem cells to terminally differentiated cells through a continuum of gene expression changes. This continuum can be treated as a high-resolution sequence of molecular events that are specific to the cell fate acquisition process (Fig. 2). Within this continuum, it will be possible to detect a set of transcription factors (TFs) that drive gene expression changes. The algorithm of the invention prioritizes and orders these TFs based on 4 main features:
  • TFs Once TFs have been identified, target networks from each of them will be screened for annotated interactions. If the interactors of two TFs show a significant degree of overlap but are involved in opposing interactions (e.g., one TF is reported to inhibit these interactors, the other one is reported to activate them), they will be flagged for incompatibility within the same screen, thus providing a more rational way to arrange combinations.
  • the algorithm of the invention can take advantage of regression-based gene- gene correlation networks and existing PPI Ns to identify molecules that act as transducers and receptors in a signalling pathway that is upstream of the TFs and is also changing along the trajectory. Once candidate receptors have been identified, their ligands can be inferred and included in the combinatorial screen.
  • small molecule-protein interaction networks overlaid on the PPINs, and drug- transcriptome correlation databases whose signature can be interrogated along a trajectory, can also be included in Daedalus for their prioritization and inclusion in the screening.
  • the CombiCult platform can be improved by greatly extending its multiplexing and resolution capabilities.
  • the implementation in W02004031369 is limited to the identification of one cell differentiation at a time, which means that several other possibilities that may have emerged in culture - such as alternative cell fates - remain unexplored.
  • I will design and implement a system to tag (“barcoding”) single cells using viral transduction.
  • barcoding is achieved through a unique, short, expressed DNA sequence that functions as a Unique Condition Tag (UCT) delivered through a lentiviral vector, which stably integrates in cells in culture (Fig. 3).
  • UCT Unique Condition Tag
  • drivers As cells go through different splits and are treated with different molecules/TFs/growth factors (hereafter referred to as “drivers”), they will be tagged by lentiviral vectors bearing a barcode unique to the condition. By using viral, integrating tags at low doses - so that cells do not become saturated at every transduction cycle - cells can be uniquely barcoded in every condition, and the UCT stays within each cell, tracking the condition in which they were inserted.
  • candidate drivers and their corresponding vectors are added to the culture media, which can be refreshed to remove viral vectors after transduction according to the needs of the protocol. Then, cells are detached, resuspended, pooled together and split in different plates for the second split.
  • scRNA-seq has been already deployed in similar ways to track guide RNA constructs in CRISPR screens (Dixit et al. 2016. Cell 167, 1853-1866) or lentivirus-mediated TF transduction (Joung et al. 2023. Cell 186, 209-229).
  • complex studies using sequential genome editing (“scarring”) for lineage tracing at the single cell level have been successfully carried out (Spanjaard et al. Nat Biotechnol . 2018 June ; 36(5): 469-473), providing a first analytical framework to connect different events in a single experiment.
  • This virtuous cycle allows the algorithms of the invention to become more and more precise, learning from their own experimental results, and paves the way for a supervised learning framework that, in the long run, can predict with high precision the molecules required for any type of differentiation.
  • ground truth i.e., data resulting from a known intervention
  • LM Language Model
  • Different types of LMs have been successfully deployed in studies of viral evolution and antibody evolution (Hie et al. 2023.
  • transdifferentiation can be driven by overexpressing specific transcription factors, as has been observed various transdifferentiation experiments:
  • fibroblasts into induced neurons (iNs).
  • - NeuroDI alone or in combination with other factors can also convert fibroblasts into neurons.
  • iCMs induced cardiomyocyte-like cells
  • Endoderm cells - FoxA2 and Hnfla can convert fibroblasts into hepatic cell types.
  • - MyoD is a classic example that can convert fibroblasts into skeletal muscle cells.
  • the method of the invention permits the generation of a database which can improve the starting parameters for an experiment designed to identify TFs suitable for reprogramming a cell. Therefore, the invention provides a method for selecting the starting TFs in a method according to the preceding aspects of the invention, comprising the steps of: compiling a database of TFs and cell transdifferentiating results; and selecting from the database a set of TFs or combinations of TFs for use in a method for determining the conditions for reprogramming a cell.
  • the invention also provides a method for improving a database of TFs and cell transdifferentiating results, comprising using the database to select TFs as in the preceding embodiment, and inputting the results of the methods for determining the conditions for reprogramming a cell into the database, thus providing further data points.
  • the method according to the invention provides: a database of TFs (for example transcription factors) useful for reprogramming cells; at least one computer processor; a memory in operative communication with the processor, the memory comprising instructions configuring the processor to:
  • GNN gene regulatory networks
  • the database preferably comprises gene expression data, such as RNA-seq data.
  • Determining a candidate transcription factor comprises interrogating a gene regulatory graph to identify the set of optimal transcription factors to differentiate any given cell type from any given starting cell.
  • a machine-learning model can be used to generate a metric calculation as a function of the gene regulatory graph.
  • Examples of a metric calculation include a criticality algorithm, which may be configured for time-series RNA-seq data; see Oh et al., Biomed Res Int. 2013;2013:203681. doi: 10.1155/2013/203681. Epub 2013 Mar 24.
  • the invention further provides a method for determining optimal transcription factors cell reprogramming, comprising: curating, using a computing device, a gene expression database related to use of TFs in cell culture; generating gene regulatory networks from a plurality of gene expression datasets; determining a candidate optimal TF; analysing an impact of the optimal TF in cell differentiation; and outputting a set of optimal TFs.
  • Methods include CellNet (et al., Cell 2014; 158:903-15), which uses microarray gene expression data and correlation-based net-work score, the method of D’Alessio et al. (D’Alessio et al., Stem Cell Reports 2015; 5:763-75) whose method uses microarray gene expression data and an information theoretic approach (Jensen- Shannon Divergence) and Mogrify (Rackham et la., Nat Genet 2016; 48(3):331-5; PMID:26711105) which uses cap analysis of gene expression (CAGE) data and a network based score.
  • CAGE cap analysis of gene expression
  • DNA template preparation and mRNA synthesis were conducted according to HiScribeTM T7 ARCA mRNA Kit (with tailing) (NED, #E2060S) protocol.
  • Flow cytometry immunostaining For the quantification of the efficiency of the serial transfection, FACS was performed. cPPs were dissociated from the beads and fixed with 1 OOpI of 4% PFA for 15 min at RT. Two washes were apply with staining buffer (BD, #554657) before permeabilized with blocking buffer for 30 min at RT. After centrifugation, conjugated antibodies were diluted at 1 :20 in blocking buffer for 30 min on ice in dark. Following incubation, two washes were performed with DPBS. At last cells were suspended in DPBS and analyzed through FACS.
  • staining buffer BD, #554657
  • c-peptide + cells For the quantification of c-peptide + cells using primary and secondary antibodies, a standardized protocol was implemented. Initially, cells were dissociated and fixed with 100 pl of 4% PFA for 30 minutes on ice. Subsequently, a single wash with 200 pl of DPBS was carried out to remove excess fixative and cellular debris before proceeding to blocking.
  • Blocking was performed on ice for 30 minutes to minimize non-specific binding of antibodies. Following this, incubation with the primary antibody was conducted overnight at low temperature to facilitate specific binding of the primary antibody to its target antigen. The next day, the cells underwent two washes with blocking solution to remove unbound primary antibody, followed by blocking with the secondary antibody on ice for 2 hours to enable detection of the primary antibody. After two additional washes with DPBS to remove excess secondary antibody, the cells were carefully collected in FACS tubes for subsequent flow cytometry analysis to quantify the c-peptide + cells accurately.
  • Table 1 The table below summarizes antibodies and respectively dilution used.
  • FACS FACS (LSRFortessa X-20, CSB) was performed using an LSRFortessa X-20 (CSB) to assess and quantify transfected cells within the population.
  • SSC side scatter
  • FSC forward scatter
  • GFP/APC/PerCP GFP/APC/PerCP versus FSC was created for additional analysis. All FACS data were evaluated using FlowJo software.
  • CombiCult® screen i. Seeding of cells onto the beads.
  • cPPs were seeded onto CombiCult® beads.
  • the beads were coated with matrigel (dilution 1 :100; BD, #354277) for at least 2 hours at 37°C.
  • Single cells were generated from confluent cPPs with GCDR incubated for 10 min at 37°C. Cells were counted to achieve the desired seeding density, and following centrifugation, cPPs were reconstituted in their basal media plus supplements and Rock inhibitor Y27632 (15).
  • Approximately 4.0 x 10 7 cPPs (250 cells/bead) were seeded onto 160,000 beads (40,000/condition), and the mixture (cells and beads) were incubated at 37°C overnight. ii. Pool/Split process.
  • the cells were distributed onto the beads in predetermined conditions (10 conditions expressing specific TF) for four days using a split-pool process, allowing to test 10.000 thousand different combinations of TFs.
  • mRNAs-TFs transfections were performed according to the matrix followed by addition of CombiCult® specific fluorescent tags at each split ( Figure 2). Simple medium with few growth factors and known molecules was used to push cPPs into [3-cells via endocrine progenitors.
  • DMEM high glucose was supplemented with Asc (50pg), 1 % B27, EGF (50ng), FGF- 7 (50ng) and RA (50nM), while next two days 5% KOSR, T3 (1 pM) and RA (25 nM) were added to DMEM high glucose.
  • Asc 50pg
  • 1 % B27 EGF
  • FGF- 7 50ng
  • RA 50nM
  • T3 (1 pM) and RA 25 nM
  • DMEM low glucose was enriched with 10% FBS, T3 (1 M) and Alk5i (10pM). iv. Screening assay. Following the pool/split process, the beads were stained at day 11 with antibody against c-peptide, functional pancreatic [3-cells marker.
  • the beads were fixed with 4% PFA (in DBPS) at RT for 30 minutes and washed three times with DPBS for 5 minutes each. Subsequently, beads were incubated with blocking solution 10% BSA or FBS with 0.5% Triton X-100 for an hour. Primary antibody (Mouse anti- pro-lnsulin c-peptide (Millipore, #05-1109)) was diluted in blocking solution (1 :1000), added to beads and incubated at 4°C overnight. Next day, three washes were performed with DBPS for 5 min each.
  • the COPAS system By utilizing the gating thresholds established by the positive and negative controls, the COPAS system analyzed the fluorescence intensity of the beads. Through this analysis, the COPAS system identified positive hits containing reprogrammed [3-cells, specifically c-peptide + beads. c) Bead digestion and isolation of tags. The c-peptide + beads were individually digested according to Plasticell’s proprietary protocol to release the fluorescence tags accumulated by the bead during the experiment. d) Tag analysis. The released tags were analyzed using FACS and the data produced were submitted and deconvoluted by proprietary CombiCult® bioinformatics program Ariadne® . e) Ariadne® Bioinformatics.
  • Ariadne® has the capability to discern specific fluorescence signal intensities as distinct tags, thereby elucidating the pathways or combinations of four TFs responsible for reprogramming cPPs into [3-cells. Furthermore, through statistical analysis, it can rank the most effective reprogramming combinations which are subsequently validated. f) Validation. 1) The validation of the top-ranked protocols started with bead-based replication of the CombiCult® results. cPPs were seeded onto matrigel-coated beads, followed by sequential delivery of TFs over four consecutive days or simultaneous delivery of TFs in a single day, using the same media as the screening process.
  • the beads were stained with antibody against c-peptide and screened using COPAS to identify those exhibiting c-peptide signals. The most effective combinations were determined by comparing the distribution of c-peptide fluorescence signals among negative and positive controls, as well as between sequential and simultaneous TF-delivery methods. 2) Subsequently, the ranked protocols underwent validation in spheroids, a common and natural method for obtaining [3-cells. In this approach, cPPs were seeded as single cells in 10 cm dishes coated with matrigel (one dish containing 12 million cells, with two dishes per sample).
  • TFs were then delivered sequentially over four days, followed by the formation of spheroids using 24 million cells (200 cells per spheroid). Transfection was performed as described previously; for each 10 cm dish, 10 pg of mRNA was diluted in 1000 pl of mRNA buffer, and 20 pl of jetMESSENGER® was added to the mix. Media was changed regularly until day 11 , as for the reprogramming experiments.
  • spheroids were stained with antibody against c-peptide for immunofluorescence analysis, while others were dissociated into single cells and stained with c-peptide to quantify through FACS the reprogramming efficiency as a measure of c-peptide expression, Additionally, another subset of spheroids was used to measure the secretion of c-peptide upon stimulation with elevated glucose levels.
  • Glucose-stimulated insulin secretion (GSIS) assays are frequently employed in research to evaluate the functional competence of differentiated and reprogrammed [3-cells. These assays involve exposing these cells to different glucose concentrations, typically low glucose (2 mM) followed by high glucose (20 mM), to assess their insulin secretion response to glucose stimulation. C-peptide secreted in low and high glucose conditions was collected and quantified using an ELISA assay (Ultrasensitive C-peptide ELISA, Mercodia #10-1141- 01).
  • Example 1 Reprogramming of adult pancreatic cells to beta cells
  • pancreatic exocrine cells were exposed to different combinations of the transcription factors Pdx1 , Ngn3, MafA, Nkx6.1 , Nkx2.2, Isl1 , NeuroDI , Rfx6, Pax4 and Pax6, using a split and pool approach.
  • Cells were screened for expression of cd49a and c- peptide, or insulin.
  • CD49a is a marker for c-peptide expressing cells. See Fig. 5.
  • T lymphocytes are fundamental actors of cellular adaptive immunity, as they use their T-Cell Receptor (TCR) to recognize foreign/pathological antigens and initiate a powerful response.
  • TCR T-Cell Receptor
  • the maturation of T lymphocytes happens in the thymus, where they upregulate different molecules that correlate with their functions.
  • CD8+ T cells display cytotoxic activity, actively killing cells that positively interact with their TCR through their antigens; CD4+ cells secrete molecules that modulate the activity of other immune cells in the environment of diseased tissue, and regulatory T cells fine-tune the responses from either population in order to avoid a potentially harmful exaggerated T cell-mediated immune response.
  • CD8+ T cells have been successfully employed in cancer immunotherapy, harnessing their cytotoxic ability via immune-checkpoint inhibitors or by adoptive cell therapy with chimaeric antigen receptor (CAR-T), the cell therapy field at large has not yet managed to consistently differentiate CD4+ and regulatory T lymphocytes for therapeutic uses (Montel- Hagen et al. Cell Stem Cell 2019 - doi: 10.1016). These represent a great opportunity for the creation of ATMPs as they play a critical role not only in the organism’s defence against cancer, but also and especially in the regulation (or in the emergence) of autoimmune diseases and immunodeficiencies. In fact, the use of CD4+ and regulatory T lymphocytes in therapy is still far from being a reality (Hippen et al. Front Immunol 2022 - doi: 10.3389), owing to the difficulties in expanding them or generating them from stem cell precursors.

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Abstract

L'invention concerne une méthode de détermination des conditions requises pour la reprogrammation d'un premier type de cellule en un second type de cellule, qui comprend les étapes suivantes : l'exposition séparée d'une première pluralité d'unités de test comprenant chacune une cellule à différents facteurs de transcription ou combinaisons de facteurs de transcription, et l'étiquetage des cellules pour indiquer l'exposition à ces facteurs de transcription ou combinaisons de facteurs de transcription ; le regroupement de la pluralité d'unités de test, et la subdivision du regroupement d'unités de test pour former une seconde pluralité d'unités de test ; l'exposition séparée de la deuxième pluralité d'unités de test à différents facteurs de transcription ou à des combinaisons de facteurs de transcription ; l'identification du type de cellule des cellules dans les unités de test et la déconvolution des étiquettes pour identifier l'identité, la séquence et la chronologie des facteurs de transcription auxquels les cellules ont été exposées ; éventuellement, l'utilisation des données de l'étape précédente pour informer le choix des facteurs de transcription utilisés dans l'étape initiale, et la répétition de l'opération si nécessaire ; et l'identification d'une cellule du second type de cellule, et la déconvolution des étiquettes pour ainsi identifier l'identité et la séquence de facteurs de transcription nécessaires pour reprogrammer une cellule du premier type de cellule en une cellule du second type de cellule.
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