Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/38—Vitamins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/15—Transforming growth factor beta (TGF-β)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/39—Steroid hormones

Definitions

• the present invention relates to populations of cells that may be used as medicaments.
• the invention also relates to methods of preparing cells for use as medicaments.
• the invention further relates to methods for selecting mesenchymal stem cell (MSC)-derived cells for therapeutic uses, and to methods of treatment using MSC-derived cells.
• MSC mesenchymal stem cell
• MSCs have been proposed as therapeutic agents for use in a wide range of applications, including tissue repair and regeneration, and amelioration of conditions such a graft-versus- host disease.
• the means by which MSCs achieve their therapeutic effects in vivo are incompletely understood, but are generally believe to include differentiation to produce new cells that contribute to replacement tissues, trophic effects that stimulate repair by neighbouring cells, and suppression of the local immune response.
• MSC-based treatments One factor that has limited the uptake of MSC-based treatments is the ability to generate therapeutically effective numbers of cells. Many treatments require large numbers of cells, that are most effectively produced by long periods of cell culture. However, it is known that properties associated with the therapeutic uses of MSCs can change significantly over time in culture. For example, the capacity of MSCs to differentiate into multiple phenotypes (multipotency), a key characteristic of stem cells, is gradually lost during prolonged cell culture, while other therapeutically relevant properties may be retained. Accordingly, prolonged culture, of the sort desirable to produce large numbers of cells, appears to be incompatible with the retention of properties that contribute to the therapeutic activities of MSCs in vivo, or with the generation of homogenous populations of the sort needed for robust cell-based medicines.
• a population of cells enriched for MSC-derived cells that are:
• the population of cells for medical use defined by the first aspect of the invention are able to stimulate trophic repair, as discussed in more detail elsewhere in the specification. These populations of cells for medical use may be referred to as “a population of cells of the invention”, and constituent cells of such populations as “cells of the invention”.
• a population of cells of the invention may be free from (or substantially free from) cells that are: CD90 + ; CD105 + ; CD45 ; and have high TIMP-1 secretion and high MMP13 gene expression.
• the medicament may be used in promoting tissue repair, and/or in promoting therapeutic immunosuppression.
• a method of preparing cells for use as a medicament comprising:
• a medicament of the invention may be free from (or substantially free from) cells that are: CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and high MMP13 gene expression.
• a method of selecting an MSC- derived cell for therapeutic use comprising:
• a method in accordance with the third aspect of the invention may optionally further comprise a step of selecting a cell for use in a therapeutic application requiring a capacity for phenotypic differentiation if the cell exhibits high levels of both MMP13 gene expression and TIMP-1 protein secretion.
• the methods of the third aspect of the invention may be put into practice by selecting populations of cells that have the desired characteristics. Examples of suitable techniques, including technique for enrichment of the desired cell types, are described elsewhere in this disclosure.
• a method of treatment comprising providing a therapeutically effective amount of cells that are:
• the subject may be one in need of tissue repair, and/or in need of therapeutic immunosuppression.
• a pharmaceutical composition comprising a population of cells enriched for MSC-derived cells that are:
• a pharmaceutical composition of the invention may be free from (or substantially free from) cells that are: CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and high MMP13 gene expression
• FIG. 1 Growth and phenotypic characteristics of MSCs from four patients
• A The number of populations doublings reached after each passage using MSCs from each patient were recorded until growth ceased (for PN242 data were collected until passage 30, when the cells continued to grow).
• B The population doubling time is shown up to passage 17 for all four patients.
• D The percentage of cells expressing MSC markers CD90 and CD105 and haematopoietic stem cell markers CD34 and CD45 were determined by Fluorescence Activated Cell Sorting. As discussed further elsewhere, the cells of the invention demonstrate a characteristic combination of being CD90 and CD105 positive, and CD45 negative.
• MSCs from passages 2-16 were seeded onto polyglycolic acid scaffolds before being induced to undergo chondrogenic differentiation. Each image shows replicate samples from each patient at each passage that was investigated.
• A-D The relationship between dry weight or glycosaminoglycans (GAG) content of tissue engineered cartilage constructs and the population doublings of MSCs at the time of tissue engineering are shown for each of the patients. For all the graphs, each point shows the mean value of multiple tissue engineering replicates made using cells from one patient at a single passage.
• GAG glycosaminoglycans
• E The relationship between dry weight of tissue engineered cartilage constructs and the passage number of MSCs at the time of tissue engineering are shown for all four patients. Each point is the result for a single replicate sample.
• A Ingenuity pathway analysis identified FOXM1 and four other master regulators genes (see Figure S5) that vary with increasing passage number.
• FOXM1 data from transcriptomics analysis shows inhibition of gene expression with increasing passage number. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• B Ingenuity pathway analysis of the integrated transcriptomics and proteomics data set predicts inhibition of five out of six of the identified downstream regulators in the FOXM1 canonical pathway.
• C Ingenuity pathway analysis identified multiple genes and proteins that mapped to the search terms “Cell Movement”, Cell Migration” or “Wound Healing”. Most of these identified genes and proteins did not vary significantly over passage as determined by ANOVA. The most highly expressed genes and proteins are listed for each of the search terms.
• CXCL12 data from transcriptomics analysis shows continuous gene expression with increasing passage number. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• CXCL12 data from proteomics analysis shows continuous protein secretion with increasing passage number. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• FIG. 7 Gene and protein markers of the in vitro MSC ageing process
• the MMP13 gene was selected as a marker of early passage cells which is lost with ageing of MSCs in vitro whilst secretion of the TIMP-1 protein was selected as a marker of MSCs that is independent of in vitro ageing.
• A MMP13 data from transcriptomics analysis shows decreasing gene expression with increasing passage number. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• MMP13 data from qPCR analysis shows decreasing gene expression with increasing passage number. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• TIMP-1 data from proteomics analysis shows continuous protein secretion with increasing passage number.
• TIMP-1 data from ELISA analysis shows continuous protein secretion with increasing passage number.
• Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• E ELISA analysis of TIMP-1 secreted by MSC/collagen scaffold constructs shows continuous protein secretion with increasing passage number for fresh constructs but reduced secretion from constructs that have been freeze-thawed under conditions that reduce their viability. This pattern of high TIMP-1 secretion and low MMP13 gene expression is characteristic of the cells of the invention.
• Figure 8 (related to Figure 3).
• Figure 9 (related to Figure 3). Osteogenic differentiation of MSCs from four patients at multiple passages
• FIG. 8 MSCs from each patient across a range of passages were induced to undergo osteogenic differentiation in vitro and scored as shown in Figure 8.
• A-D The relationship between osteogenic score and the cumulative population doublings of MSCs are shown for each of the patients. For all the graphs, each point shows the mean value of multiple replicates using cells from one patient at a single passage.
• E The relationship between the osteogenic score and the passage number of MSCs are shown for all four patients. Each point is the result for a single replicate sample. For all graphs, the line represents a linear model of degree 1 fitted to the points whilst the spearman rank correlation coefficient and its significance is shown in the top right corner.
• FIG 10 (related to Figures 3).
• MSCs from each patient across a range of passages were induced to undergo adipogenic differentiation in vitro and scored as shown in Figure S3.
• A-D The relationship between adipogenic score and the cumulative population doublings of MSCs are shown for each of the patients. For all the graphs, each point shows the mean value of multiple replicates using cells from one patient at a single passage.
• E The relationship between the adipogenic score and the passage number of MSCs are shown for all four patients. Each point is the result for a single replicate sample. For all graphs, the line represents a linear model of degree 1 fitted to the points whilst the spearman rank correlation coefficient and its significance is shown in the top right corner.
• Ingenuity pathway analysis identified FOXM1 (See Figure 6) and four other master regulators genes that vary with increasing passage number.
• A Relationship between MYOC data from transcriptomics analysis and increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics data set. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• B Relationship between NUPR1 data from transcriptomics analysis and increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics data set. Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• C Relationship between VEGF data from transcriptomics analysis and increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics data set.
• Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• D Relationship between PTGER2 data from transcriptomics analysis and increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics data set.
• Each bar is the mean ⁇ SEM for results using MSCs from each of the four patients.
• the present invention is based, at least in part, upon the inventors’ finding, disclosed for the first time in this document, that MSCs cultured in vitro exhibit a selective loss of multipotent potential, but not trophic activity or immunosuppressive activity, prior to senescence.
• This finding, and the observation that many of the therapeutic uses of MSCs and MSC-derived cells arise as a consequence of the trophic or immunosuppressive activity of such cells, rather than through phenotypic differentiation of these cells opens a new possibility of providing therapeutic uses of cultured cells that would previously have been discounted from such applications. That extensively cultured cells can be used therapeutically in this manner confers a significant benefit, in that it allows the generation of much larger numbers of therapeutically useful cells than would previously have been thought possible.
• the inventors have identified a characteristic panel of markers that allows those cells that retain trophic or immunosuppressive activity, but are not capable of phenotypic differentiation, to be distinguished from those cells that retain capacity for phenotypic differentiation (either with or without trophic or immunosuppressive activity).
• This characteristic expression profile (cells that are: CD90 + ; CD105 + ; CD45 ; have high TIMP-1 secretion and low MMP13 gene expression) has not been identified before. Its identification here provides significant advantages for medical use of MSC-derived cells in practice.
• this marker profile avoids the need for selection based upon criteria, such as passage number, that are associated with significant failings.
• criteria such as passage number
• these include the impact of variations among individuals in the point at which this change occurs (relevant for both allogeneic and autologous cell sources), variation in rates of cell division within cell cultures, and reliance on considerations, such as passage number, that are not relevant in the case of modern bioreactor-based culture conditions.
• Such cells may be CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and high MMP13 gene expression, in contrast to the cells of the invention (that are CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and low MMP13 gene expression).
• the present invention identifies for the first time that:
• MSC-derived cells retain the ability to exert these therapeutic mechanisms after extensive cell expansion
• MSCs are used for a wide range of clinical applications for tissue regeneration (e.g. bone and cartilage; cardiovascular disease) as well as for disease modification, including haematological disease, graft-versus-host disease and inflammatory diseases. Some of these clinical approaches are based on differentiation whilst others depend on trophic or immunoregulatory functions.
• Populations of cells of the invention For the present purposes, the considerations below may be taken as applicable to populations of cells according to the first aspect of the invention, as well as to populations of cells selected in the methods of the second aspect of the invention, or for use in the methods of treatment of the fourth aspect of the invention.
• Cells of the invention may be autologous cells, or may be allogeneic cells.
• the cells of the invention may not have the capacity for multipotent differentiation.
• the skilled person will be aware of many methods by which multipotency, or absence of multipotency, may be determined, including (but not limited to) those discussed in the Examples.
• the therapeutically useful MSC-derived cells referred to in the various aspects of the present invention exhibit a characteristic expression pattern in respect of a number of markers. These include cell surface markers (CD90, CD105, and CD45) and functional markers (TIMP-1 and MMP13).
• CD90 also known as Thy-1, is an N-glycosylated glycophosphatidylinositol (GPI) anchored cell surface protein that has a single V-like immunoglobulin domain.
• GPI glycophosphatidylinositol
• CD105 also known as endoglin, is a type I membrane glycoprotein. It is located on the cell surface, where it functions as part of the TGF-b receptor complex. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure are characteristically CD105T
• CD45 also known as protein tyrosine phosphatase receptor type C (PTPRC)
• PPRC protein tyrosine phosphatase receptor type C
• CD45 ⁇ is a type I transmembrane protein involved in a number of cell signalling pathways.
• PPRC protein tyrosine phosphatase receptor type C
• cell surface markers including CD90, CD105 and CD45 may be investigated by any suitable means known to those skilled in the art.
• cell surface markers may be labelled and detected by appropriate reagents such as antibodies specific for the cell markers of interest.
• Antibody labelling approaches of this sort can be used in combination with cell selection techniques of the sorts further described below.
• a cell may be considered positive (“+ve” or “ + ”) for a particular cell surface marker (such as CD90 or CD105) if, after appropriate immunofluorescence labelling, it has a level of fluorescence that is greater than 90% of cells treated with corresponding control antibodies, such as isotype-matched control antibodies.
• a particular cell surface marker such as CD90 or CD105
• a cell may be considered negative for a particular cell surface marker (such as CD45) if, after appropriate immunofluorescence labelling, it has a level of fluorescence that is less than 10% of cells treated with corresponding control antibodies, such as isotype-matched control antibodies.
• a particular cell surface marker such as CD45
• TIMP-1 also known as TIMP metallopeptidase inhibitor 1
• TIMP metallopeptidase inhibitor 1 is a member of the tissue inhibitor of metallopeptidases family. It is a glycoprotein that functions as an inhibitor of matrix metalloproteinases.
• the therapeutically useful MSC-derived cells described in this disclosure characteristically have high TIMP-1 protein secretion.
• MMP13 matrix metallopeptidase 13, also known as collagenase 3
• collagenase 3 is a metalloproteinase enzyme involved in the breakdown of extracellular membrane components. It is encoded by the MMP13 gene in humans.
• the therapeutically useful MSC- derived cells described in this disclosure characteristically have low MMP13 gene expression. The relevance of this marker in allowing the selection of cell populations that have trophic and immunosuppressive activity, but not the capacity for phenotypic differentiation, has not previously been reported.
• this marker is key in distinguishing cells of the invention (which are: CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and low MMP13 gene expression) from other populations of cells that do not offer the same therapeutic advantages, but retain capacity for phenotypic differentiation (which are: CD90 + ; CD105 + ; CD45 ; and have high TIMP-1 secretion and high MMP13 gene expression).
• the cells of the invention are characterised as having high TIMP-1 protein secretion.
• TIMP-1 protein expression may be investigated using any suitable technique for the assessment of protein secretion known to those skilled in the art.
• TIMP-1 protein expression may be investigated using an enzyme linked immunoassay (ELISA) approach.
• ELISA enzyme linked immunoassay
• Examples of an appropriate ELISA for TIMP-1 secretion that may be used to determine whether or not cells of interest are positive for TIMP-1 secretion in the manner required of cells of the invention is described further in the Examples.
• the cells of the invention are characterised as having low MMP13 gene expression.
• MMP13 gene expression may be assessed by investigating levels of transcripts indicative of MMP13 expression within a cell.
• MMP13 gene expression may be assessed by MMP13 specific reverse transcription PCR. A particularly suitable method based upon this technique is described further in the Examples.
• a population of cells may be identified as having high TIMP-1 secretion on the basis of the quantity of this protein secreted by a set number of cells over a set period of time.
• Suitably high secretion of TIMP-1 protein may be indicated by the secretion of 100ng/ml (or more) of TIMP-1 from cells (initially plated at a density of 2.25 million cells/well) in a 24 hour period. Further details of suitable conditions that may be used in such an assessment are set out in the Examples, in connection with the study reported in Figure 7E.
• a population of cells may be identified as having low MMP13 gene expression on the basis of comparison to a suitable baseline.
• a housekeeping gene such as Tata binding protein may provide a suitable baseline.
• a population of cells may be characterised as having low MMP13 gene expression if these cells demonstrate a level of MMP13 expression relative to a housekeeping gene (such as Tata binding protein) that is half or less than half of the relative expression by freshly isolated MSCs.
• Suitable freshly isolated MSCs for comparison purposes may be derived from a source that is appropriately matched to that of the cells of the invention. Comparison of folds of expression (whether of MMP13 or suitable housekeeping genes) may be carried out using the methods described further in the Examples.
• a population of cells in accordance with the first aspect of the invention is enriched for the presence of therapeutically useful MSC-derived cells, as compared to naturally occurring cell populations.
• Cells prepared in a method of the second aspect of the invention are also enriched for the presence of such cells, and the selection of cells in accordance with the third aspect of the invention may give rise to enriched populations of cells in the same manner.
• the therapeutically useful MSC-derived cells to be enriched within a population may be identified with reference to their characteristic expression of markers (i.e. CD90 + , CD105 + , CD45-; high TIMP-1 protein secretion; and low MMP13 gene expression).
• markers i.e. CD90 + , CD105 + , CD45-; high TIMP-1 protein secretion; and low MMP13 gene expression.
• enriched as used in the context of cell populations. Enrichment may be achieved by positive selection of desired cells (i.e. those with the characteristic expression of markers) or by elimination of undesired cells.
• enrichment approaches may make use of culture conditions that favour the proliferation of cells with desired traits (such as the characteristic expression of markers discussed herein), and/or conditions that diminish the viability of cells that lack desired traits. Cells grown in such conditions may be monitored to ensure that the desired population are enriched.
• cell culture conditions that can be used to enrich populations of therapeutically effective MSC-derived cells in accordance with the invention may include the use of 10% foetal calf serum, and supplementation with 5ng/ml fibroblast growth factor (FGF2).
• a method of preparing cells for use as a medicament in accordance with the second aspect of the invention may comprise a step of selecting cells with reference to one or more of the recited markers (CD90 + ; CD105 + ; CD45-; high TIMP-1 secretion; and low MMP13 gene expression), and thus enriching the MSC-derived cells for the specified population of cells.
• a suitable selection step may comprise selection based on low MMP13 gene expression, a marker that the inventors have found to be particularly useful in identifying cells that retain trophic activity without potential for phenotypic differentiation.
• an enriched population of MSC-derived cells may comprise at least 80% of cells that are positive for CD90 and CD105, and 10% or less of cells that are positive for CD45.
• an enriched population of cells may comprise at least 70% cells having the recited characteristic marker profile (i.e. CD90 + ; CD105 + ; CD45 ; high TIMP-1 secretion; and low MMP13 gene expression).
• an enriched population of cells may comprise at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% cells having the recited characteristic marker profile.
• An enriched population of cells may consist of substantially 100% cells having the recited characteristic marker profile (i.e. CD90 + ; CD105 + ; CD45-; high TIMP-1 secretion;
• a population of cells in accordance with the first aspect of the invention, or selected in a method of the second aspect of the invention, may substantially consist entirely of cells having the recited characteristic marker profile (i.e. CD90 + ; CD105 + ; CD45 ; high TIMP-1 secretion; and low MMP13 gene expression).
• a population of cells in accordance with the first aspect of the invention, or selected in a method of the second aspect of the invention, may be free, or substantially free, from cells which are: CD90 + ; CD105 + ; CD45 ; and have high TIMP-1 secretion and high MMP13 gene expression.
• Cell enrichment or cell selection may be practiced by any suitable method, including, but not limited to, those discussed herein.
• cells of the invention may retain therapeutically effective levels of trophic or immunoregulatory activity for up to 30 passages.
• a population of cells of the invention may comprise cells that have been subject to at least 5 passages in culture, at least 10 passages in culture, at least 15 passages in culture, at least 20 passages in culture, at least 25 passages in culture, or at least 30 passages in culture.
• a method in accordance with the second aspect of the invention may comprise culturing the cells for at least 5 passages in culture, at least 10 passages in culture, at least 15 passages in culture, at least 20 passages in culture, at least 25 passages in culture, or at least 30 passages in culture.
• the populations of cells of the first aspect of the invention, the medicaments produced from cells prepared in accordance with the second aspect of the invention, the cells selected by the methods of the third aspect of the invention, and the methods of treatment of the fourth aspect of the invention are all suitable for therapeutic use.
• the therapeutic use may be a use to promote tissue repair, or use to promote therapeutic immunosuppression. Further details of these embodiments are set out below.
• Tissue repair Medicaments medical uses or methods of treatment of the invention may be used to promote tissue repair.
• the requirement for promotion of tissue repair may arise as a result of any condition causing tissue injury.
• the requirement for promotion of tissue repair may arise as a result of a condition selected from the group consisting of: osteoarthritis; myocardial infarction; meniscus cartilage injury (such as torn meniscus); ligament injury (such as torn ligament); injuries to the skin; and soft tissue injury.
• the tissue repair may be promoted by promoting therapeutic trophic activity.
• the tissue repair may ameliorate pathology associated with a diseased or damaged joint. This may be assessed by appropriate means, such as histological examination (of biopsies or experimentally treated joints) or suitable imaging techniques.
• the tissue repair may comprise treatment of a damaged tissue to promote tissue repair by improving the rate of tissue repair and/or improving the quality of repaired tissue that results from treatment.
• the promotion of tissue repair may be readily assessed with reference to a suitable control.
• the cells, methods of treatment or medical uses of the invention may be capable of promoting tissue repair by at least 10% as compared to suitable controls.
• the cells, methods of treatment or medical uses of the invention may be capable of promoting tissue repair by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to suitable controls.
• the cells, methods of treatment or medical uses of the invention may be capable of promoting tissue repair by 100% or more as compared to suitable controls.
• the tissue repair may comprise treatment of a damaged tissue selected from the group consisting of: cartilage; cardiovascular tissue; bone; and soft tissue, such as skin.
• the tissue repair may comprise treatment of a condition selected from the group consisting of: treatment of torn cartilage, such as treatment of torn meniscal cartilage; treatment of: osteoarthritis; myocardial infarction; meniscus cartilage injury (such as torn meniscus); ligament injury (such as torn ligament); injuries to the skin; and soft tissue injury.
• torn cartilage such as treatment of torn meniscal cartilage
• meniscus cartilage injury such as torn meniscus
• ligament injury such as torn ligament
• injuries to the skin and soft tissue injury.
• trophic activity of MSC-derived cells may contribute to tissue repair.
• a medical use or method of treatment employing a population of cells or medicament of the invention may be for use in tissue repair by the stimulation of trophic activity.
• Such uses may be contrasted with those applications that have previously been described in which MSCs or MSC-derived cells achieve their therapeutic utility via phenotypic differentiation (i.e. through differentiation to yield replacement cells that contribute to tissue repair).
• the cells of the invention have trophic activity that is retained from the MSCs from which they are derived.
• the medical uses and methods of treatment of the invention particularly those for use in tissue repair, may make use of the trophic activity of such cells. Indeed cells of the invention may be selected for their trophic activity (and absence of capacity for phenotypic differentiation).
• Trophic activity may be considered as the capacity by which, following implantation, MSCs or MSC-derived cells are able to induce neighbouring cells to secrete active molecules that contribute to tissue repair or regeneration.
• Trophic repair may occur as a result of the production by the MSCs, or MSC-derived cells, of large amounts of growth factors and other mediators.
• Trophic activity has been shown to contribute to tissue repair or regeneration in the treatment of stroke, myocardial infarction, and meniscal cartilage repair, among other examples.
• the methods of the second or third aspect of the invention may further comprise a step of assessing the cells to investigate their trophic activity.
• Suitably cells to be used as a medicament may be selected based upon their trophic activity.
• the populations of cells in accordance with the first aspect of the invention may be enriched for MSC-derived cells that have trophic activity.
• the presence or absence of trophic activity may be assessed by any suitable methods known to those skilled in the art. Suitable methods may investigate the ability of cells to ameliorate pathology of diseased or damaged joints.
• the trophic activity of cells such as cells of the first aspect of the invention, or prepared in a method of the second aspect of the invention, may be assessed by testing their capacity to integrate separate regions of cartilage. Suitably the capacity to promote such integration may be demonstrated in vitro. Alternatively, or additionally, such capacity may be demonstrated in vivo.
• Therapeutic applications that can benefit from the trophic activity of cells or medicaments, or medical uses or methods of treatment, of the invention correspond to those that will benefit from promotion of tissue repair.
• the cells of the invention also have immunosuppressive activity that is retained from the MSCs from which they are derived.
• the medical uses and methods of treatment of the invention may make use of the cells’ immunosuppressive activity, and may be for use in therapeutic immunosuppression.
• the therapeutic immunosuppression may be employed in the treatment of a disease selected from the group consisting of: haematological disease; graft-versus-host disease; and alloreactive immune disease, autoimmune disease, or inflammatory disease.
• Therapeutic immunosuppression using the cells, methods of treatment or medical uses of the invention may be utilised in the treatment of patients that have received stem cell grafts, such as grafts of haematopoietic stem cells. Patients receiving such grafts may be suffering from a haematological disease.
• treatment with cells or medicaments of the invention may be able to reduce the recipient’s immune response, and thus alleviate the response to an allogeneic graft.
• a cell, medicament or method of treatment is accordance with the invention is for use in treatment of a disease by immunosuppression of the immune response.
• the methods of the second or third aspect of the invention may further comprise a step of assessing the cells to investigate their immunosuppressive activity.
• Suitably cells to be used as a medicament may be selected based upon their immunosuppressive activity.
• the populations of cells in accordance with the first aspect of the invention may be enriched for MSC-derived cells that have immunosuppressive activity.
• Immunosuppressive activity of cells or a medicament of the invention may be compared to that of a suitable control.
• the cells or medicaments of the invention may be able to achieve at least a 10% suppression of immune activity as compared to controls, when assessed in a suitable assay.
• the cells or medicaments of the invention may, for example be able to achieve at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, or at least a 95% suppression of immune activity as compared to suitable controls.
• the cells or medicaments of the invention may be able to achieve 100% suppression of immune activity as compared to suitable controls.
• the immunosuppressive activity of cells may be assessed by testing their capacity to inhibit T-cell proliferation.
• immunosuppressive activity is indicated by the ability to inhibit T-cell proliferation by at least 80%, consistent with the results set out in Figure 4I.
• the third aspect of the invention provides a method by which suitable MSC-derived cells can be selected for therapeutic applications. This selection is undertaken with reference to the nature of the therapeutic application, and also the expression of certain markers by the MSC- derived cells.
• the methods of the third aspect of the invention allow a distinction to be made between cells to be used in a therapeutic application that requires a capacity for phenotypic differentiation, and cells that are to be used in a therapeutic application that does not require a capacity for phenotypic differentiation.
• Therapeutic applications that require a capacity for phenotypic differentiation will be those in which the therapeutic activity of MSC-derived cells is achieved by the ability of these cells to differentiate and generated new cells that provide a therapeutic effect.
• Examples of such applications include in vitro tissue engineering, in which MSCs are induced to differentiate and thereby to produce articular cartilage or bone, and the use of MSCs for transplantation into cartilage lesions or bone defects for the in situ generation of replacement tissues.
• therapeutic applications that do not require MSC-derived cells to have a capacity for phenotypic differentiation in order to achieve a therapeutic effect. It will be appreciated that therapeutic applications that make use of trophic effects to induce tissue repair do not require a capacity for phenotypic differentiation, since it is trophic factors produced by MSCs or MSC-derived cells that mediate therapeutic activity of this sort. Similarly, it will be recognised that therapeutic applications that make use of the immunosuppressive effects of MSC-derived cells do not require a capacity for phenotypic differentiation. Examples of therapeutic applications that make use of trophic effects or immunosuppressive effects to achieve the required therapeutic activity are described elsewhere in the present specification.
• the populations of cells of the first aspect of the invention, cells prepared in accordance with the second aspect of the invention, and methods of treatment of the fourth aspect of the invention are also highly suitable for use in such applications that do not require a capacity for phenotypic differentiation or multipotency.
• compositions including the cells of the invention including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
• the pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient.
• the composition includes at least one additional therapeutic agent.
• pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
• a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
• a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
• the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
• the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
• Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
• Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001 to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
• the formulations can include aqueous solutions.
• the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
• active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
• the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
• chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
• the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
• Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
• the desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
• the cells and compositions may be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous.
• immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
• Peripheral blood derived immunoresponsive cells or their progeny e.g., in vivo, ex vivo or in vitro derived
• localized injection including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration.
• a therapeutic composition e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell
• it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
• Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
• the cell populations are administered parenterally.
• parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
• the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
• compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
• sterile liquid preparations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
• Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
• Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
• carriers can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
• Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
• a suitable carrier such as a suitable carrier, diluent, or excipient
• the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
• compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
• antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
• Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
• Formulations of cells of the invention may be free, or substantially free, from cells which are: CD90 + ; CD105 + ; CD45-; and have high TIMP-1 secretion and high MMP13 gene expression. Doses and therapeutically effective amounts of cells of the invention
• the therapeutically effective amount of cells of the invention may be selected with reference to the nature of the disorder to be treated, and also with reference to the severity of the disease in an individual requiring treatment. Considerations such as the age and weight of the recipient may also influence the selection of an appropriate dose.
• a therapeutically effective amount of cells of the invention may be provided in a single incidence of treatment, or by multiple incidences of treatment.
• a suitable number of therapeutically effective MSC-derived cells for use in accordance with the invention may be calculated on the basis of the number of cells administered per site of injury.
• a dose of between 10 and 20 million cells per knee may prove therapeutically effective. This guidance may be adjusted depending on the organ to be treated, or for larger or smaller sites of injury.
• Bone marrow-derived mesenchymal stromal cells are defined as stem cells in part because of their capacity for multipotent differentiation, however more recently they have been shown also to have trophic and immunoregulatory properties that may be important for promoting tissue repair.
• MSCs bone marrow-derived mesenchymal stromal cells
• MSCs multipotent mesenchymal stem cells
• MSCs may support tissue repair through mechanisms that do not directly relate to their multipotential differentiation capacity (Prockop, 2007).
• Caplan has described MSCs as having “trophic” capacity by which, following implantation, they induce neighbouring cells to secrete active molecules, for example in the treatment of stroke, myocardial infarction or in meniscal cartilage repair (Caplan and Dennis, 2006).
• Trophic repair is most likely mediated through the production by MSCs of large amounts of growth factors and other mediators (Caplan and Correa, 2011; Caplan and Dennis, 2006; Kuroda et al., 2011; Prockop, 2009; Tolar et al. , 2010).
• MSCs meniscal cartilage repair based on the trophic properties of MSCs
• a second mechanism of tissue repair that is independent of multipotent differentiation is the ability of MSCs to suppress immune responses by a range of mechanisms including downregulation of T cell proliferation (Dickinson et al., 2017; Keating, 2012; Spaggiari et al., 2007; Tolar et al., 2010; Uccelli et al., 2006; Uccelli et al., 2007).
• This important property of MSCs has been used clinically to support the engraftment of donated haematopoetic cells and to prevent graft versus host disease (Lazarus et al., 2005; Tolar et al., 2010).
• ageing of MSCs in vitro will provide a framework in which to understand the relationship between different aspects of their biology, by identifying those functional properties that are transient and those which are core properties that are retained throughout the lifespan of the cells.
• the aim of this study was therefore to determine the relative hierarchical importance of differentiation potential and trophic repair/immunoregulation by determining the rate at which these functions decline with increasing MSC ageing in vitro.
• Bone marrow was collected from patients undergoing arthroplastic surgery for treatment of traumatic knee injury. All patients gave informed consent and the study was performed in full accordance with local ethics guidelines (Southmead Research Ethics Committee Ref 078/01). Table S1 shows the patient characteristics. All four were male with a mean age of 49 years (range, 38-70 years) at the time of operation. MSCs were isolated from each bone marrow by plastic adhesion and grown under standard culture conditions until they failed to proliferate any further. MSCs from PN241 and PN242 continued to proliferate for a larger number of passages than MSCs from PN251 and PN264, with PN242 cells showing no sign of growth arrest even at passage 30 ( Figure 1A and Table S1).
• Figure 1B shows the MSC population doubling time (PDT) at each passage for each patient up to passage 17.
• PDT MSC population doubling time
• the tri-lineage differentiation potential of MSCs falls with increasing passage MSCs from selected passages of each patient were tested for their chondrogenic potential in a three-dimensional cartilage tissue engineering assay, with the amount of engineered cartilage measured as the dry weight of tissue and quality of the cartilage measured as the glycosaminoglycans content expressed as a % of dry weight.
• the typical macroscopic appearance of tissue engineered cartilage over a range of passages for MSCs from each of the patients can be seen in Figure 2, clearly demonstrating a reduction in the average size of cartilage constructs when produced using late passage MSCs. This macroscopic observation was supported by quantitative analysis.
• MSCs are also able to suppress immunity through inhibition of T-Cell proliferation and other mechanisms.
• RNA from the undifferentiated MSCs of all four patients at each passage was reserved. Based on growth and differentiation characteristics we selected RNA from passages P1 , P5, P10 and P15 for gene array comparison. For each of these passages we also collected conditioned medium for proteomic comparison. The genomic and proteomic data were combined and analysed for patterns of change in related genes and proteins. The methodological approach to transcriptomic and proteomic analysis is illustrated in Figure 5A. Data structure was appraised via principal component analysis (PCA). A score plot of the first two principal components is shown in Figure 5B. The changes captured over passage are accounted for in PC2 whilst PC1 captures considerable variation between patients which can be stratified in two groups (shown with blue and orange ellipses).
• PCA principal component analysis
• I PA core analysis, overlaid with the global molecular network within the software resulted in the identification of a number of canonical pathways, functions and upstream regulators found to be significantly over-represented within this list and therefore linked to the loss of multipotenial differentiation capacity of the cells.
• upstream regulators predicted to be deactivated were the prostaglandin receptor PTGER2, and members of the VEGF family, whilst upstream regulators that were also predicted to be positively activated over passage included the proliferation regulator NUPR and the cytoskeleton regulator MYOC. Quantitative data from our transcriptomic analyses and the associated I PA predictions of changes in the downstream effectors of these regulators, are shown in Figure S5, however none of the changes and predicted downstream effects were as clear-cut as for FOXM1.
• Regulators of cell migration and wound healing can be linked to the trophic properties ofMSCs
• the I PA analysis outlined above demonstrated downregulation of the FOXM1 canonical pathway with increasing passage/loss of multi-potent differentiation and continuous expression of CXCL12 and other cell migration and wound healing genes and proteins with increasing passage.
• genes and proteins that may not be part of canonical pathways or gene/protein families, but that can be used as specific markers of cellular ageing in vitro. Such markers would aid in comparison of studies of cells from one laboratory to another or in determining the functionality of an MSC population being used for therapeutic purposes. We therefore analysed the gene array and protein data to identify candidate markers.
• MMP13 matrix metalloproteinase 13
• IGF Binding protein 5 IGF Binding protein 5
• MSCs expressing high levels of the MMP13 gene whilst secreting abundant TIMP-1 could potentially be used for therapeutic processes involving either phenotypic differentiation or trophic/immunoregulatory repair.
• MSCs expressing low levels of the MMP-13 gene whilst secreting abundant TIMP-1 could only be used for processes involving trophic/immunoregulatory repair, but they could not be used reliably for those processes involving differentiation.
• CXCL12 (also known as SDF-1) was found to be associated with all three of our search terms related to trophic repair and its gene and protein levels were maintained even at very late passage numbers, indicating a potential role for CXCL12 in MSC-mediated trophic repair. This observation is consistent with previous studies which have demonstrated its critical role in MSC-mediated induction of spinal cord repair (Stewart et al., 2017) and myocardial repair (Dong et al., 2012), as well as enhancing nerve cell survival in vitro (Chalasani et al., 2003) and mediating trophism between endothelial cells and tumour cells (Rao et al., 2012).
• TIMP-1 is an inhibitor of MMP13 and as well as other metalloproteinases.
• Muraglia et al (Muraglia et al., 2000) also showed a loss of multipotent differentiation with passage, but in their experiments, osteogenesis was retained with early loss of adipogenesis. However, they were working with clonal MSC cell-lines whereas the work reported here and by Yang et al investigated the whole MSC population.
• Haynesworth et al first described the unique cytokine expression pattern of MSCs (Haynesworth et al., 1996) and they demonstrated a reduction in cytokine production after stimulating differentiation using dexamethasone.
• Caplan and Dennis coined the term “trophic effect” which they defined as “those chemotactic, mitotic, and differentiation- modulating effects which emanate from cells as bioactive factors that exert their effects primarily on neighbouring cells and whose effects never result in differentiation of the producer cell” (Caplan and Dennis, 2006). They cited, as a typical example of this effect, the support provided by MSCs in the bone marrow niche for growth and differentiation of haematopoetic cells.
• MSCs providing trophic support in a range of cell therapy settings including the treatment of stroke, myocardial infarction and meniscal cartilage regeneration.
• Numerous other studies have gone on to describe the importance of MSC- induced trophic repair (Caplan and Correa, 2011; Caplan and Dennis, 2006; Kuroda et al. , 2011; Prockop, 2009; Tolar et al., 2010).
• MSC-induced trophic repair Caplan and Correa, 2011; Caplan and Dennis, 2006; Kuroda et al. , 2011; Prockop, 2009; Tolar et al., 2010.
• We exploited the trophic effects of MSCs in devising a new method of treating fresh meniscal cartilage tears using a stem cell/collagen-scaffold implant to promote integration of the damaged tissue.
• T-cell proliferation is a critical component of their suppressive activity (Dickinson et al. , 2017; Keating, 2012; Spaggiari et al., 2007; Tolar et al., 2010; Uccelli et al., 2006; Uccelli et al., 2007).
• Human T-cells can be strongly stimulated to proliferate using a combination of anti-CD3 and anti-CD28 antibodies (Verhagen and Wraith, 2014).
• MSCsenchymal Stem Cells was coined by Caplan (Caplan, 1991), who went on to describe MSCs as “An injury Drugstore” (Caplan and Correa, 2011) and more recently advocated a change in their name to “Medicinal Signaling Cells” (Caplan, 2017).
• Other studies have questioned the definition of MSCs as stem cells because of the lack of rigorous confirmatory biological evidence (Bianco et al., 2008; Javazon et al., 2004; Keating, 2012; Kuroda et al., 2011; Prockop, 2009), with all of these studies calling for more experimental data before reaching a conclusion on the nomenclature.
• Bone marrow plugs were collected from the femoral heads of patients undergoing total hip replacement. All patients gave their informed consent and the study was carried out according to local ethical guidelines (North Bristol NHS Trust Research Ethics Committee). Patient details can be seen in Table S1. Cells were suspended in stem cell expansion medium consisting of low glucose Dulbecco’s Modified Eagles Medium (Sigma) supplemented with 10% (v/v) Foetal Bovine Serum (FBS, Thermo Scientific Hyclone, Loughborough, UK), 1% (v/v) Glutamax (Sigma) and 1% (v/v) Penicillin/Streptomycin (Sigma).
• stem cell expansion medium consisting of low glucose Dulbecco’s Modified Eagles Medium (Sigma) supplemented with 10% (v/v) Foetal Bovine Serum (FBS, Thermo Scientific Hyclone, Loughborough, UK), 1% (v/v) Glutamax (Sigma) and 1% (v/v) Penicillin/Streptomycin (S
• the serum batch was selected to promote the growth and differentiation of MSCs (Kafienah et al., 2007a).
• the medium was also supplemented with 10 ng/ml FGF-2 (Peprotech).
• FGF-2 Peprotech
• This growth factor has been previously shown to enhance the MSC proliferation rate in vitro (Bianchi et al., 2003; Solchaga et al., 2005), to retain MSCs as undifferentiated cells during proliferation (Kafienah et al., 2006; Martin et al., 1999) and to enhance chondrogenic differentiation when the FGF-2 expanded MSCs are subsequently exposed to differentiation conditions (Bianchi et al., 2003; Solchaga et al., 2005).
• the cell suspension was separated from any bone in the sample by repeated washing with media.
• the cells were centrifuged at 500 g for 5 minutes and the supernatant/fat removed.
• the resulting cell pellet was resuspended in medium, and then plated at a seeding density of between 1.5-2.0x10 5 nucleated cells per cm 2 .
• These flasks were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Four days were allowed before the first medium change and then the medium was changed every other day until adherent cells reached 90% confluence and were ready for passaging.
• the MSCs were harvested using 0.25% trypsin-EDTA (Invitrogen), pooled, counted and then divided into different centrifuge tubes for reseeding and further growth, for immediate use in measurement of % integration of meniscal cartilage, for storage in liquid nitrogen for subsequent use in differentiation protocols as well as for genomic and proteomic analysis.
• the cells for each patient were passaged continuously without freezing, until growth arrest, defined as no detectable increase in cell number between passages (See Table S1).
• the total number of harvested MSCs was determined.
• the first cell harvest after seeding of fresh bone marrow was taken as passage 0.
• the number of cells reseeded at the start of Passage 1 was used as the baseline for calculation of the first population doubling value at the end of passage 1.
• Downstream analyses of the MSCs were undertaken from passage 1 onwards.
• the number of Population doublings (PDs) was calculated using the following formula:
• PDs [log(Number of Harvested MSCs) log(Number of seeded MSCs)]/log(2)]
• the PD for each passage was calculated and added to the PD of the previous passages to generate data for Cumulative PD at each Passage.
• MSCs 100,000 cells from each patient at each of passages 1, 5, 10 and 15 were suspended in a 1 :500 dilution of Zombie (Biolegend), a live/dead cell dye, and incubated for 20mins in the dark. Nonspecific antigens were then blocked by incubating the cells at room temperature for 1 hour in 1% (wt/vol) BSA (Sigma-Aldrich), 5% (vol/vol) FCS (Sigma-Aldrich), and 10% (vol/vol) human serum (Sigma-Aldrich).
• the cells were washed by centrifugation in three volumes of PBS, and the cell pellet was suspended in 10OmI of a primary antibody solution containing 20-100pg/ml of antibody in blocking solution.
• All the primary antibodies were fluorescent-labelled mouse anti-human IgGs: anti-CD105-fluorescein isothiocyanate (FITC), anti-CD90-phycoerythrin (PE), anti-CD45-PE were from R&D Systems); anti-CD34-FITC was from BD Bioscience; lgG1-FITC and lgG1-PE isotype controls were from R&D Systems.
• PGA polyglycolic acid
• chondrogenic differentiation medium consisting of DMEM containing 4500 mg/L glucose (Sigma-Aldrich), supplemented with 10 ng/ml transforming growth factory (TQR-b3; R&D Systems), 100 nM dexamethasone, 80 mM ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 1% (v/v) Penicillin/Streptomycin (all from Sigma-Aldrich), 1% insulin-transferrin-selenium-G (ITS) and 2 mM Glutamax-I (both from Invitrogen).
• the medium was further supplemented with 10 pg/ml bovine pancreatic insulin (Sigma-Aldrich) until the end of culture.
• the constructs were incubated at 37°C for a total of 35 days on a rotating platform and medium was changed every three days.
• Cartilage constructs were freeze-dried and weighed at the end of the 35-day tissue engineering period.
• the extracellular matrix was fully solubilised by overnight digestion with 2 mg/ml bovine pancreatic trypsin (Sigma-Aldrich) which was then boiled for 15 min to inhibit the action of the enzyme (Dickinson et al., 2005).
• remaining undigested scaffold material was freeze-dried, weighed and subtracted from the original dry weight.
• the amounts of proteoglycan in the digests was measured as sulphated glycosaminoglycan (GAG) using a dimethylmethylene blue (Sigma-Aldrich) colorimetric assay (Handley and Buttle, 1995).
• Meniscal cartilage cylinders (5.0 mm in diameter and 3.0 mm thick) were harvested from the avascular (white zone) of the ovine menisci using a dermal biopsy punch. They were rinsed and incubated with phosphate buffered saline (PBS; Invitrogen Ltd, Paisley, UK) containing 10% (v/v) Penicillin/Streptomycin (Sigma-Aldrich) and 1% (v/v) 250pg/ml Amphotericin B (Sigma-Aldrich) for 20 minutes.
• PBS phosphate buffered saline
• Penicillin/Streptomycin Sigma-Aldrich
• Amphotericin B (Sigma-Aldrich) for 20 minutes.
• Viability of the fibrocartilage discs was maintained by culture in basic medium consisting of low-glucose DMEM with 10mM Hepes buffer (Sigma), 1% (v/v) Penicillin/Streptomycin, non-essential amino acids (NEAA; Sigma), 1% (v/v) Glutamax and 10% amphotericin B at 37 ° C in a 5% CO2 environment.
• the explants were used in the integration experiments within 3 days of culture.
• Collagen scaffolds (Ultrafoam Collagen Sponge; Bard, UK, www.barduk.com) were cut into 6mm diameter discs and seeded with human MSCs at a concentration of 1 x 10 6 cells/cm 2 .
• the suspension was loaded drop wise onto the scaffold placed in ultra-low attachment wells of a 24-well plate (Corning®, Acton, USA). After 4 hours, 1.5 ml of expansion medium containing 10 ng/ml FGF-2 was added and changed daily. Seeded scaffolds were incubated for 48 hours at 37°C in an orbital shaker at 50 rpm.
• Sandwich constructs of two ovine meniscal cartilage discs interposed with a seeded scaffold were assembled as previously described (Whitehouse et al., 2017) using skin clips and cultured in vitro in ultra-low attachment 6-well plates in expansion medium with 10 ng/mL FGF- 2 for 7 days followed by culture in an integration medium consisting of high glucose DMEM containing 10% (v/v) FBS, 1% (v/v), Glutamax, 1% (v/v) Penicillin/Streptomycin, insulin and ascorbate-6-phosphate (50pg/ml; Sigma) for 33 days. The medium was replenished twice every week. The constructs were incubated at 37°C on a rotating platform throughout the culture period. At the end of culture, the constructs were prepared for histological analysis by fixation in 10% (v/v) neutral buffered formalin.
• Histomorphometry was carried out using a method that we developed and characterized in previous studies (Pabbruwe et al., 2009; Pabbruwe et al., 2010a; Whitehouse et al., 2017). Fixed constructs were dehydrated and paraffin embedded. Samples were cut into 4pm sections and stained with hematoxylin and eosin (H&E) for the study of morphological details. All histological sections were scanned using a Leica Aperio slide scanner and histomorphometric analysis was performed under blind conditions, using ImageScope software (Leica). Two perpendicular sections, one at the edge and another one at the center of each construct were used. For each section, the entire length of the implant/meniscus interface was measured, as well as the length of any areas of integration at the interface. The repair index was then determined as:
• PBMCs Human peripheral blood mononuclear cells
• PBMCs were then stimulated with 3.75pg/ml anti-human CD3 (HIT3a) and 2pg/ml anti-human CD28 (CD28.2) (both from Fisher Scientific Affymetrix eBioscience, Cheshire, UK) and co-cultured with MSCs from 4 individual donors at passages 1, 5, 10 and 15 for 72 hours.
• HIT3a anti-human CD3
• CD28.2 2pg/ml anti-human CD28
• the T-cell proliferation profile for each population was analysed by flow cytometry following exclusion of non-viable cells stained with 7-amino- actinomycin D (7-AAD; BD Biosciences).
• RT-qPCR Real-time quantitative PCR
• MMP13 mRNA and for the housekeeping gene Tata Binding Protein were performed using the CellsDirectTM One-Step qRT-PCR Kit (ThermoFisher), with which reverse transcription and PCR amplification were performed in the same reaction tube.
• Primers specific for MMP13 Hs00942584_m1
• TBP Hs00427621_m1
• ThermoFisher TaqMan® ThermoFisher TaqMan®. The reaction was started by synthesising cDNA at 50°C for 15min, followed by 2min at 95°C to denature RNA-cDNA hybrids and deactivate reverse transcriptase.
• the thermal cycling program consisted of 50°C for 15 minutes, 95°C for 2 minutes, and 40 two-step cycles of 95°C for 10s and 60°C for 30s.
• MMP13 expression relative to TBP was determined at each of 4 time points for each MSC sample and the results normalised to the time (passage 2), which was taken as a Fold- expression of 1.0.
• TIMP-1 protein was measured in the secretome of MSCs using the Quantikine® ELISA Kit for human TIMP-1 (R&D Systems). MSCs were seeded into 6-well plates at 2.25 million cells/well (3 replicates ) and cultured in 2ml of DMEM for 24 h. The medium was then replaced with 1 ml of phenol-free culture medium for a further 24h, after which the secretome was collected and assayed at appropriate dilution using the ELISA kit.
• T ranscriptomics was performed by the Centre for Genomic Research on mRNA extracted from the mRNA of all four patients at P1, P5, P10 and P15.
• MSCs RNAprotect Cell Reagent
• the cells were stored at -80°C until the complete set of samples from all donors and time points had been collected.
• RNA was then extracted from selected time points using the RNeasy Plus Mini Kit (Qiagen), according to the manufacturer’s instructions.
• the concentration of RNA in the extract was determined using a NanoDrop 2000 spectrophotometer (Thermo). Extracted RNA was stored at -80°C prior to analysis.
• Ribosomal RNA depletion was performed using the Ribo-ZeroTM H/M/R Kit (lllumina) and RNASeq libraries were then prepared using the NEB Next Ultra Directional RNA Library Prep Kit (lllumina). Paired-end sequencing of the RNASeq libraries was performed by the lllumina HiSeq4000 platform using V4 chemistry.
• Proteomics was performed on secretome prepared from the MSCs of all four patients at P1, P5, P10 and P15.
• MSCs were harvested at the end of each passage, 1x10 6 cells were isolated and resuspended in 4 mL of serum-free, phenol red-free DMEM (Sigma) supplemented with 4500 mg/L glucose, 1% (v/v) Glutamax (Sigma), 1% (v/v) P/S (Sigma). and 2 mM Glutamax-I for 24 hours at 37°C.
• the conditioned medium recovered at the end of the incubation was harvested from each flask and stored at -80°C prior to analysis.
• the beads were re-suspended in 80mI_ of 25 mM ambic and 5mI_ of 1%(w/v) Rapigest in 25 mM ambic (Waters Limited, Hertfordshire, UK) was added.
• the samples were heated at 80°C for 10 minutes and then reduced by the addition of 5 pL of dithiothreitol (DTT, 9.2mg/mL in 25mM ambic) and heating at 60°C for 10mins.
• DTT dithiothreitol
• iodoacetamide 33mg/mL in 25mM ambic
• Porcine trypsin (sequencing grade, Promega) (1 pg) was added and the sample was incubated at 37°C overnight on a rotary mixer.
• the digests were acidified by the addition of 1 pL of trifluoracetic acid (TFA) and incubated at 37°C for 45 minutes. Samples were then centrifuged at 17,200 x g for 30mins and supernatants transferred to 0.5 mL low-binding tubes. They were centrifuged for a further 30mins and 10pL transferred to total recovery vials for LC-MS analysis.
• TFA trifluoracetic acid
• the trapping column was then set in-line with an analytical column (EASY-Spray PepMap RSLC C18, 75pm x 50cm, 2pm packing material, 100A) and the peptides eluted using a linear gradient of 96.2% A (0.1 %[v/v] formic acid): 3.8% B (0.1% [v/v] formic acid in water: acetonitrile [80:20] [v/v]) to 50% A:50% B over 90min at a flow rate of 300nL min -1 , followed by washing at 1% A : 99% B for 5 minutes and re-equilibration of the column to starting conditions.
• an analytical column EASY-Spray PepMap RSLC C18, 75pm x 50cm, 2pm packing material, 100A
• the column was maintained at 40°C, and the effluent introduced directly into the integrated nano-electrospray ionisation source operating in positive ion mode.
• the mass spectrometer was operated in DDA mode with survey scans between mlz 350-2000 acquired at a mass resolution of 60,000 (FWHM) at mlz 200.
• the maximum injection time was 100ms, and the automatic gain control was set to 3e6.
• the 16 most intense precursor ions with charges states of between 2+ and 5+ were selected for MS/MS with an isolation window of 2 mlz units.
• the maximum injection time was 45ms, and the automatic gain control was set to 1e5. Fragmentation of the peptides was by higher-energy collisional dissociation using a normalised collision energy of 30%.
• Oxidation of methionine was selected as a dynamic modification and carbamidomethyl cysteine as a fixed modification. One missed cleavage was permitted.
• the Mascot search returned 2,594 proteins at 2.17% FDR (psms above homology).
• a 1%FDR was set and 2,366 proteins were exported as an .xml file (FDR type: distinct psms) and imported into Progenesis and peptides assigned to proteins. Protein quantification was based on averaging the individual abundances for each protein per donor at each passage and comparing proteins that were differentially expressed between the 4 passages.
• RNAseq data was acquired as described above.
• the raw Fastq files were trimmed for the presence of lllumina adapter sequences using Cutadapt version 1.2.1, option -O 3. Reads were further trimmed using Sickle version 1.200 with a minimum window quality score of 20. Reads shorter than 10 base pairs after trimming were removed. Sequence quality metrics were assessed using FastQC version 0.11.4. No samples were removed.
• Sequence data were aligned to the human genome version GRCh38 from NCBI using Bowtie2 version 1.1.2 with recommended parameters (Langdon, 2015). Gene level count data were generated from the Bowtie2 alignments using htseq-count version 0.9.0.
• the R library DESeq2 was used to produced rlog transformed count data. These were filtered to remove genes with less than 1 average count.
• Statistical analyses were performed in R version 3.4.4 and graphical representations were done using the R package ggplot2.
• TE, GAG, and osteogenesis and adipogenesis scores were compared over passage by calculating the non-parametric Spearman correlation embedded in the function contest within the stats package in R. Osteogenesis and adipogenesis capabilities were measured with semi quantitative data based on image analysis. These were translated into integers 0 to 1 to undertake the calculation.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Developmental Biology & Embryology (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Hematology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Rheumatology (AREA)
• Virology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicinal Chemistry (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=68538920

Family Applications (1)

Country Status (7)

Families Citing this family (2)

Citations (3)

• 2019
  • 2019-10-02 GB GB201914235A patent/GB201914235D0/en not_active Ceased
• 2020
  • 2020-10-02 EP EP20788864.5A patent/EP4038179B1/en active Active
  • 2020-10-02 KR KR1020227014379A patent/KR20220092880A/ko active Pending
  • 2020-10-02 WO PCT/GB2020/052439 patent/WO2021064424A1/en not_active Ceased
  • 2020-10-02 AU AU2020361080A patent/AU2020361080A1/en active Pending
  • 2020-10-02 JP JP2022520605A patent/JP7710242B2/ja active Active
  • 2020-10-02 US US17/761,565 patent/US20220389386A1/en active Pending

Patent Citations (3)

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events