Marine invertebrate collagen use

Abstract

The present invention relates to methods of isolating and / or culturing a population of mesangiogenic progenitor cells (MPCs) or cells of the endothelial lineage, wherein the methods comprise the use of marine invertebrate collagen. The invention also relates to compositions comprising MPCs and marine invertebrate collagen and medical uses of such compositions, and medical devices and kits comprising the same.

Description

[0001] MARINE INVERTEBRATE COLLAGEN USE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods of isolating and / or culturing a population of mesangiogenic progenitor cells (MPCs) or cells of the endothelial lineage, wherein the methods comprise the use of marine invertebrate collagen. The invention also relates to compositions comprising MPCs and marine invertebrate collagen and medical uses of such compositions, and medical devices and kits comprising the same.

[0004] BACKGROUND

[0005] Following their identification, mesenchymal stromal / stem cells (MSC) generated substantial interest due to their differentiation plasticity and hematopoietic supportive function. In particular, it was thought that MSCs may hold great potential in the treatment of bone and cartilage defects in patients. However, subsequent investigations showed that MSCs have relatively low engraftment efficiency and only a transient benefit in the mitigation of the symptoms of congenital and acquired conditions in patients. Indeed, many clinical and pre-clinical studies showed disappointing outcomes, especially in relation to efficacy endpoints achieved, including a lack of a long-lasting and consolidated repair of the defects treated. One of the proposed reasons of those unsatisfactory results was absent of insufficient vascularisation of the newly-formed patient tissue derived from MSCs.

[0006] In addition, it was also found that in both bone marrow and adipose tissue, MSCs are very rare (typically from 0.001 to 0.01% of total nucleated cells) and so those cells need to be expanded in vitro in order to harvest a clinically relevant number of cells. Such expansion can take several weeks of in vitro cell culture and represents a significant limitation to the application of MSC in the manufacturing of medical products, not only in terms of costs of production, but through the increased risk of infection and tumorigenic transformation of the cultured MSCs.

[0007] A new bone marrow-derived multipotent cell type called the mesangiogenic progenitor cell (MPC) has recently been characterised in humans. That cell demonstrates mesengenic and angiogenic potential and can be readily differentiated into tri-potent MSCs (i.e. MSCs having osteogenic, adipogenic, and chondrogenic potential) or induced toward vasculogenic (i.e. blood vessel-forming) cell lineages. MPCs therefore represent a promising culture-initiating source of cells for the production of clinical grade MSCs and / or vascular cell types. Furthermore, MPCs are found at frequencies one to two logs higher than other MSC progenitors described in human bone marrow.

[0008] However, MPCs are very prone to spontaneously differentiate towards the MSC lineage in culture. Indeed, small modifications of the culture conditions can trigger uncontrolled mesengenic differentiation, thereby depleting the MPC population and consequently reducing or removing the mesengenic and vasculogenic potential of that cell population.

[0009] For example, when mammalian collagen (e.g. bovine PureCol®) is used as coating agent or as matrix scaffold for MPC culture, such collagen leads to induction of MPC cell stress and mesengenic differentiation of the MPCs towards the MSC lineage. In addition, the use of mammalian collagen also promotes calcification of long-term cultures. Thus, during the isolation and culturing of MPCs for use in clinical applications, one of the most important goals is the maintenance of the MPCs in their undifferentiated state.

[0010] Accordingly, there is a need to develop methods of isolating, culturing, and scaffolding MPCs for therapeutic applications involving the use of MPCs that overcome the problems present in the prior art.

[0011] Surprisingly, the applicant has found that isolating and culturing MPCs in the presence of marine invertebrate collagen leads to improved maintenance of MPCs in a quiescent and undifferentiated state, with a significant reduction in unwanted differentiation of those cells when compared to isolation and culturing in the presence of mammalian collagen products. Specifically, the applicant has found that isolating and culturing MPCs in the presence of marine invertebrate collagen significantly reduces differentiation of those cells into cells of the mesenchymal stromal cell (MSC) lineage, including osteogenic cell types. Moreover, the applicant has demonstrated that by culturing in combination with marine invertebrate collagen, MPCs can specifically and rapidly respond to chondrogenic stimuli, osteogenic stimuli, or a combination of those stimuli, in order to produce newly formed, specialised tissue. Accordingly, using the methods of the present invention it is possible to obtain clinically relevant populations of MPCs and / or populations with a greater frequency of cells that have therapeutically useful differentiation potential.

[0012] In addition, the applicant has found that, when appropriately induced, MPCs cultured in the presence of marine invertebrate collagen (as 3D cell scaffolds) are advantageously able to promote formation of new blood vessels, demonstrating improved results when compared to fibronectin.

[0013] DESCRIPTION OF THE INVENTION

[0014] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be understood by those of ordinary skill in the art that embodiments of the present invention may be practiced without these specific details while still remaining within the scope of the claims.

[0015] In a first aspect, the present invention provides a method of selectively isolating a population of mesangiogenic progenitor cells (MPCs), wherein the method comprises culturing a bone marrow-derived sample in the presence of marine invertebrate collagen and a culture medium.

[0016] In a second aspect, the present invention provides method of culturing or scaffolding a population of mesangiogenic progenitor cells (MPCs), wherein the method comprises culturing or scaffolding MPCs in the presence of marine invertebrate collagen and a culture medium.

[0017] "Marine invertebrate" refers to invertebrate animals that live in marine habitats. Marine invertebrates make up much of the macroscopic life in the oceans and have a large variety of body plans. They can be categorised into over 30 phyla, including Acoela, Annelida, (polychaetes and sea leeches), Brachiopoda (marine animals that have hard "valves" (shells) on the upper and lower surfaces), Bryozoa (also known as moss animals or sea mats), Chaetognatha (commonly known as arrow worms), Cephalochordata (represented in the modern oceans by the lancelets), Cnidaria (such as jellyfish, sea anemones, and corals), Crustacea (including lobsters, crabs, shrimp, crayfish, barnacles, hermit crabs, mantis shrimps, and copepods), Ctenophora (also known as comb jellies, the largest animals that swim by means of cilia), Echinodermata (including sea stars, brittle stars, sea urchins, sand dollars, sea cucumbers, crinoids, and sea daisies), Echiura (spoon worms), Gnathostomulids (slender to thread-like worms, with a transparent body that inhabit sand and mud beneath shallow coastal waters), Gastrotricha (often called hairy backs, found mostly interstitially in between sediment particles), Hemichordata (includes acorn worms, solitary worm-shaped organisms), Kamptozoa (goblet-shaped sessile aquatic animals, with relatively long stalks and a "crown" of solid tentacles, also called Entoprocta), Kinorhyncha (segmented, limbless animals, widespread in mud or sand at all depths, also called mud dragons), Loricifera (very small to microscopic marine sediment-dwelling animals), Mollusca (including shellfish, squid, octopus, whelks, Nautilus, cuttlefish, nudibranchs, scallops, sea snails, Aplacophora, Caudofoveata, Monoplacophora, Polyplacophora, and Scaphopoda), Myzostomida (small marine worms which are parasitic on crinoids or "sea lilies"), Nemertinea (also known as "ribbon worms" or "proboscis worms"), Orthonectida (among the simplest of multi-cellular organisms), Phoronida (marine animals that filterfeed with a lophophore (a "crown" of tentacles and build upright tubes of chitin to support and protect their soft bodies), Placozoa, (small, flattened, multicellular animals around 1 millimetre across and the simplest in structure), Porifera (sponges, multicellular organisms that have bodies full of pores and channels allowing water to circulate through them), Priapulida (marine worms that live marine mud), Pycnogonida (also called sea spiders), Sipunculida (also called peanut worms, is a group of bilaterally symmetrical, unsegmented marine worms), Tunicata (also known as sea squirts or sea pork, are filter feeders attached to rocks or similarly suitable surfaces on the ocean floor), some flatworms of the classes Turbellaria and Monogenea, Xenoturbella (bilaterian animals that contains only two marine worm-like species), Xiphosura (including a large number of extinct lineages and only four recent species in the family Limulidae, which include the horseshoe crabs).

[0018] There are multiple methods for 'isolating', or 'purifying' collagenous material from the anatomical milieu of marine invertebrates and such methods are well known to the skilled person. For example, collagenous material can be purified from jellyfish by acid extraction, whereby different anatomical parts of the jellyfish are bathed in an acidic solution. 'Bathing', or 'bathed', refers to the process of incubating the jellyfish in the acid solution for a sufficient amount of time in order to liberate the collagenous material. An alternative method for purification of collagenous material is enzyme extraction, whereby the jellyfish is incubated with at least one proteolytic enzyme for a sufficient amount of time and under conditions that favour the degradation of the anatomical milieu in order to liberate the collagenous material. The exact temperature, pH and incubation time of the enzyme extraction method will vary depending on the proteolytic enzyme used. The most suitable conditions will be well known to the skilled person. By way of non-limiting example, the enzyme pepsin can be incubated with jellyfish material under acidic conditions in order to liberate the collagenous material. In an embodiment of the second aspect, the method further comprises isolating the population of MPCs.

[0019] By "isolating a population of MPCs" we include the meaning of obtaining a population of cells comprising cells having the CD73ne9CD90ne9CD45dimCD31bri9htphenotype and demonstrating mesengenic and angiogenic potential in vitro. Isolation of MPCs may be carried out on bone marrow-derived samples. Protein markers identifying MPCs can be detected using techniques known in the art such as immunofluorescence-based approaches using antibodies directed to the protein markers, confocal microscopy, flow cytometry, or Western blotting.

[0020] By "scaffolding" we include the meaning of combining cells with a defined and temporary solid or semi-solid 3D structure. That 3D structure is able to interact with the cells such that it provides attachment and / or differentiation support to those cells.

[0021] In some embodiments of the first and second aspects, the marine invertebrate collagen is in its atelo form.

[0022] By "atelo form" we include the meaning of a low-immunogenic derivative of collagen obtained by removal of N- and C-terminal telopeptide components, which are known to induce antigenicity in humans. Telopeptides are generally removed by treatment of collagen with type I pepsin.

[0023] In some embodiments of the first and second aspects, the marine invertebrate collagen is in its telo form.

[0024] By "telo form" we include the meaning of a collagen extracted under acidic conditons to produce a soluble collagen that includes telopeptides.

[0025] In some embodiments of the first and second aspects, the marine invertebrate collagen is thiolated.

[0026] The term "thiolated" is intended to refer to a marine invertebrate collagen which has been reacted with a thiol, resulting in the introduction of a -SH group, or 'thiol' group. In some embodiments of the first and second aspects, the marine invertebrate collagen is cross-linked.

[0027] In the context of the present invention, the term 'cross-linked' refers to the linkage of two independent collagen molecules via a covalent bond. Preferably, the collagen molecules to be cross-linked are in the form of collagen fibres, resulting in inter-fibril cross-linking occurring. In order to create the cross-linked thiolated jellyfish collagen, a 'cross-linking agent' or 'cross-linker' may be used. The term 'cross-linking agent' or 'cross-linker' refers to an agent that can, under certain conditions, form covalent linkages between two independent molecules. In the context of the present invention, a cross-linking agent is used to covalently link two independent collagen molecules. Preferably, the collagen molecules to be cross-linked are in the form of collagen fibres. Preferably inter-fibril crosslinking takes place. In some instances, the cross-linking agents are typically composed of two or more reactive functional groups linked together by a hydrocarbon chain. The two or more functional groups do not necessarily have to be the same. The length of the hydrocarbon chain can also be varied to control the distance between the functional groups. The exact length of the hydrocarbon chain in the context of the present invention is not intended to be limiting.

[0028] In some embodiments of the first and second aspect, the marine invertebrate collagen is not cross-linked.

[0029] In some embodiments of the first and second aspect, the marine invertebrate collagen is methacrylated.

[0030] By "methacrylated" we include the meaning of a marine invertebrate collagen which has been modified by reacting free amines with methacrylamide so as to introduce methacrylate groups.

[0031] In preferred embodiments of the first and second aspects, the marine invertebrate collagen is jellyfish collagen.

[0032] "Jellyfish" refers to the medusa-phase of gelatinous members of the subphylum Medusozoa, a major part of the phylum Cnidaria. Jellyfish are mainly free-swimming marine gelatinous zooplanktonic organism with umbrella-shaped bells and trailing tentacles, although a few are not mobile, being anchored to the seabed by stalks. The bell can pulsate to provide propulsion and highly efficient locomotion. The tentacles are armed with stinging cells and may be used to capture prey and defend against predators. Jellyfish have a complex life cycle: the medusa is normally the sexual phase; the planula larva can disperse widely and is followed by a sedentary polyp phase.

[0033] In some embodiments of the first and second aspects, the jellyfish collagen is from jellyfish of the class Scyphozoa. In some embodiments, the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof. In preferred embodiments, the jellyfish is Rhizostomas pulmo.

[0034] In some embodiments of the first and second aspects, the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0035] "Sponge matrix" refers to a matrix or scaffold which is soft, highly flexible, porous and hydrophilic in nature, made up of a blend of different polymers, synthetic and / or natural, with high flexibility and absorbent capacity. Such sponges can be in a micronised form and be used to absorb the extracts of the invention and release it at the surface of the human skin through contact.

[0036] "Nano-fibre electrospun matrix" refers to a matrix made of threads of polymer solutions or polymer melts up to fiber diameters in the order of some hundred nanometers. Such matrices are useful for medical purpose because they can be penetrated with cells or drug substances to treat or replace biological targets. For example, nanofibrous wound dressings have excellent capability to prevent microbial infections in wounds.

[0037] "Hydrogel" refers to a network of soluble collagen fibres that are prevented from dissociating by polymer entanglement and / or covalent cross-linking. Such polymer chains that are hydrophilic, resulting in a highly absorbent material. Hydrogels can also be formed from colloidal suspensions. The physical properties of hydrogels can be tuned depending on the route of manufacture.

[0038] In some embodiments of the first and second aspects, the marine invertebrate collagen is coated onto a tissue culture surface. In some embodiments of the first and second aspects, the marine invertebrate collagen coated onto a tissue culture surface is present at a concentration of at least lpg / cm2, at least 2pg / cm2, at least 3pg / cm2, at least 4pg / cm2, at least 5pg / cm2, at least 6pg / cm2, at least 7pg / cm2, at least 8pg / cm2, at least 9pg / cm2, at least 10pg / cm2, at least 1 lpg / cm2, at least 12pg / cm2, at least 13pg / cm2, at least 14pg / cm2, at least 15pg / cm2, at least 16pg / cm2, at least 17pg / cm2, at least 18pg / cm2, or at least 19pg / cm2, at least 20pg / cm2, at least 25pg / cm2, at least 30pg / cm2, at least 35pg / cm2, at least 40pg / cm2, at least 45pg / cm2, at least 50pg / cm2, or at least 60pg / cm2, at least 70pg / cm2, at least 80pg / cm2, at least 90pg / cm2, or at least 100pg / cm2. Preferably, the marine invertebrate collagen coated onto a tissue culture surface is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

[0039] In some embodiments of the first and second aspects, the method prevents or reduces differentiation of the population of MPCs into cells of the mesenchymal stromal cell (MSC) lineage.

[0040] In some embodiments of the first and second aspects, the method prevents or reduces spontaneous and uncontrolled differentiation of the population of MPCs into cells of the mesenchymal stromal cell (MSC) lineage.

[0041] By "cells of the mesenchymal stromal cell (MSC) lineage" we include the meaning of a cell population having the CD73bri9btCD90br'9btCD45ne9CD31ne9phenotype and demonstrating multi-lineage differentiation capability towards one or more of the ostegenic, adipogenic, or chondrogenic lineages. Protein markers that identify cells of the MSC lineage can be detected using techniques known in the art such as immunofluorescence-based approaches using antibodies directed to the protein markers, confocal microscopy, flow cytometry, or Western blotting.

[0042] Similarly, MPCs display their own characteristic expression of biological markers. For instance, MPCs lack markers seen in MSCs such as CD73 and CD166 and the MPC phenotype is also characterised by expression of CD31bri9btand CD45dim. MPCs are also equipped with unusual sub-cellular structures called "podosomes" which are sustained by integrin [32 in a heterodimer with integrin oX, oL, or oM. Protein markers that identify MPCs can be detected using techniques known in the art such as immunofluorescence- based approaches using antibodies directed to the protein markers, confocal microscopy, flow cytometry, or Western blotting.

[0043] In some embodiments of the first and second aspects, the population of MPCs comprises less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 10%, less than 15%, or less than 20% of cells of the MSC lineage. Preferably, the population of MPCs comprises less than 15% of cells of the MSC lineage, more preferably the population of MPCs comprises less than 10% of cells of the MSC lineage, most preferably the population of MPCs comprises less than 5% of cells of the MSC lineage.

[0044] In some embodiments of the first and second aspects, the culture medium is MPC- maintaining medium. In some embodiments, the MPC-maintaining medium comprises Dulbecco's Modified Eagles Medium (DMEM) and serum. In some embodiments, the DMEM is low glucose DMEM. In some embodiments, the serum is pooled human AB-type serum (PhABS) or autologous serum.

[0045] In some embodiments, the serum is at a concentration of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, or at least 30%. Preferably, the serum in the MPC maintaining medium is at a concentration of at least 1%, more preferably at least 5%, most preferably at least 10%.

[0046] In some embodiments of the first and second aspects, the MPC-maintaining medium comprises L-glutamine and / or pyruvate.

[0047] In some embodiments of the first and second aspects, the L-glutamine is at a concentration of at least 0.5mM, at least ImM, at least 2mM, at least 3mM, at least 4mM, at least 5mM, at least 6mM, at least 7mM, at least 8mM, at least 9mM, at least lOmM, at least 15mM, or at least 20mM. Preferably, the L-glutamine is at a concentration of at least 0.5mM, more preferably at least ImM, most preferably at least 2mM.

[0048] In some embodiments of the first and second aspects, the pyruvate is at a concentration of at least lOmg / L, at least 20mg / L, at least 30mg / L, at least 40mg / L, at least 50mg / L, at least 60mg / L, at least 70mg / L, at least 80mg / L, at least 90mg / L, at least lOOmg / L, at least HOmg / L, at least 120mg / L, at least 130mg / L, at least 140mg / L, at least 150mg / L, at least 160mg / L, at least 170mg / L, at least 180mg / L, at least 190mg / L, or at least 200mg / L. Preferably at least 25mg / L, more preferably at least 50mg / L, yet more preferably at least 75mg / L, most preferably at least lOOmg / L.

[0049] In some embodiments of the first and second aspects, the MPC-maintaining medium further comprises antibiotic compounds and / or antifungal compounds.

[0050] By "MPC-maintaining medium" we include the meaning of a culture medium which prevents or reduces the likelihood of MPCs differentiating to a cell type with less differentiation potential (i.e. a cell type that is less pluripotent).

[0051] In some embodiments of the first and second aspects, the method promotes maintenance of the population of MPCs in their undifferentiated state.

[0052] In some embodiments of the first and second aspects, the population of MPCs have MPC differentiation potential.

[0053] By "MPC differentiation potential" we include the meaning of the capability of a single MPC, when cultured at low-density under mesengenic-differentiating conditions, to generate a single colony (commonly defined as colony forming unit fibroblastoid or CFU-F) of at least fifty fibroblastoid cells that are characterised as MSCs in accordance with the definition above (i.e. cells having the CD73bri9btCD90br'9btCD45ne9CD31ne9phenotype and demonstrating multi-lineage differentiation capability towards one or more of the ostegenic, adipogenic, or chondrogenic lineages).

[0054] In some embodiments of the first and second aspects, the population of MPCs are capable of differentiating into cells of the endothelial cell lineage.

[0055] By "MPC vasculogenic differentiation potential" we include the meaning of the capability of a single MPC, when low-density cultured in endothelial differentiating condition that replicates the Hill's assay, to generate a single colony (commonly defined as colony forming cells in the Hill's assay: CFU-Hill) of at least fifty cells that are characterised as pre- endothelial cells following analysis using characterisational confocal microscopy, flow cytometry or Western blotting in order to detect CD31 and VEGFR-2 protein markers. In some embodiments of the first and second aspects, the MPCs are capable of differentiating into blood vessel cells, cartilage cells, muscle, and / or bone cells.

[0056] In some embodiments of the first and second aspects, after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential and / or are capable of differentiating into cells of the endothelial cell lineage is at least 5%, at least 10%, at least 15%, 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%, at least 95%, or at least 99%. Preferably, after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential is at least 70%, more preferably 80%, yet more preferably 90%, most preferably 95%.

[0057] In some embodiments of the first and second aspects, after at least 48 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential and / or are capable of differentiating into cells of the endothelial cell lineage is at least 5%, at least 10%, at least 15%, 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%, at least 95%, or at least 99%. Preferably, after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential is at least 70%, more preferably 80%, yet more preferably 90%, most preferably 95%.

[0058] In some embodiments of the first and second aspects, after at least 72 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential and / or are capable of differentiating into cells of the endothelial cell lineage is at least 5%, at least 10%, at least 15%, 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%, at least 95%, or at least 99%. Preferably, after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential is at least 70%, more preferably 80%, yet more preferably 90%, most preferably 95%.

[0059] In some embodiments of the first and second aspects, the population of MPCs are mammalian cells. In some embodiments, the mammalian cells are human cells, mouse cells, rat cells, dog cells, cat cells, cow cells, sheep cells, goat cells, horse cells, or pig cells. Preferably, the mammalian cells are human cells.

[0060] In a third aspect, the present invention provides a population of MPCs obtained by a method according to the first aspect or the second aspect.

[0061] In a fourth aspect, the present invention provides a method of obtaining a population of cells of the endothelial lineage, wherein the method comprises culturing a population of MPCs in the presence of marine invertebrate collagen and a culture medium that promotes differentiation of the population of MPCs into cells of the endothelial lineage.

[0062] In some embodiments of the fourth aspect, the cells of the endothelial cell lineage comprise blood vessel-forming cells and / or endothelial cells.

[0063] In some embodiments of the fourth aspect, the culture medium that promotes differentiation of the population of MPCs into cells of the endothelial lineage comprises Endothelial Cell Growth Media (EGM-2).

[0064] In some embodiments of the fourth aspect, the culture medium that promotes differentiation of the population of MPCs is serum-free.

[0065] In some embodiments of the fourth aspect, the culture medium that promotes differentiation of the population of MPCs comprises L-glutamine and / or pyruvate.

[0066] In some embodiments of the fourth aspect, the L-glutamine is at a concentration of at least 0.5mM, at least ImM, at least 2mM, at least 3mM, at least 4mM, at least 5mM, at least 6mM, at least 7mM, at least 8mM, at least 9mM, at least lOmM, at least 15mM, or at least 20mM. Preferably, the L-glutamine is at a concentration of at least 0.5mM, more preferably at least ImM, most preferably at least 2mM.

[0067] In some embodiments of the fourth aspect, the pyruvate is at a concentration of at least lOmg / L, at least 20mg / L, at least 30mg / L, at least 40mg / L, at least 50mg / L, at least 60mg / L, at least 70mg / L, at least 80mg / L, at least 90mg / L, at least lOOmg / L, at least llOmg / L, at least 120mg / L, at least 130mg / L, at least 140mg / L, at least 150mg / L, at least 160mg / L, at least 170mg / L, at least 180mg / L, at least 190mg / L, or at least 200mg / L. Preferably at least 25mg / L, more preferably at least 50mg / L, yet more preferably at least 75mg / L, most preferably at least lOOmg / L.

[0068] In some embodiments of the fourth aspect, the culture medium that promotes differentiation of the population of MPCs into cells of the endothelial lineage further comprises antibiotic compounds and / or antifungal compounds.

[0069] In some embodiments of the fourth aspect, the marine invertebrate collagen is in its atelo form. In other embodiments, the marine invertebrate collagen is in its telo form.

[0070] In some embodiments of the fourth aspect, the marine invertebrate collagen is thiolated.

[0071] In some embodiments of the fourth aspect, the marine invertebrate collagen is crosslinked. In other embodiments, the marine invertebrate collagen is not cross-linked.

[0072] In some embodiments of the fourth aspect, the marine invertebrate collagen is methacrylated.

[0073] In preferred embodiments of the fourth aspect, the marine invertebrate collagen is jellyfish collagen.

[0074] In some embodiments of the fourth aspect, the jellyfish collagen is from jellyfish of the class Scyphozoa. In some embodiments, the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof. In preferred embodiments, the jellyfish is Rhizostomas pulmo.

[0075] In some embodiments of the fourth aspect, the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0076] In some embodiments of the fourth aspect, the marine invertebrate collagen is coated onto a tissue culture surface. In some embodiments of the fourth aspect, the marine invertebrate collagen coated onto a tissue culture surface is present at a concentration of at least lpg / cm2, at least 2pg / cm2, at least 3pg / cm2, at least 4pg / cm2, at least 5pg / cm2, at least 6pg / cm2, at least 7pg / cm2, at least 8pg / cm2, at least 9pg / cm2, at least 10pg / cm2, at least 1 lpg / cm2, at least 12pg / cm2, at least 13pg / cm2, at least 14pg / cm2, at least 15pg / cm2, at least 16pg / cm2, at least 17pg / cm2, at least 18pg / cm2, or at least 19pg / cm2, at least 20pg / cm2, at least 25pg / cm2, at least 30pg / cm2, at least 35pg / cm2, at least 40pg / cm2, at least 45pg / cm2, at least 50pg / cm2, or at least 60pg / cm2, at least 70pg / cm2, at least 80pg / cm2, at least 90pg / cm2, or at least 100pg / cm2. Preferably, the marine invertebrate collagen coated onto a tissue culture surface is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

[0077] In some embodiments of the fourth aspect, the population of cells of the endothelial lineage are mammalian cells. In some embodiments, the mammalian cells are human cells, mouse cells, rat cells, dog cells, cat cells, cow cells, sheep cells, goat cells, horse cells, or pig cells. Preferably, the mammalian cells are human cells.

[0078] In a fifth aspect, the present invention provides a population of cells of the endothelial lineage obtained by a method according to the fourth aspect.

[0079] In a sixth aspect, the present invention provides a composition for use in medicine, wherein the composition comprises:

[0080] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of the first aspect or second aspect; or

[0081] (ii) a population of MPCs and marine invertebrate collagen.

[0082] In a seventh aspect, the present invention provides a composition for use in tissue regeneration in a subject, wherein the composition comprises:

[0083] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of first aspect or second aspect; or

[0084] (ii) a population of MPCs and marine invertebrate collagen. In an eighth aspect, the present invention provides a method of promoting tissue regeneration in a subject comprising administering to the subject a composition comprising:

[0085] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of the first aspect or second aspect; or

[0086] (ii) a population of MPCs and marine invertebrate collagen.

[0087] In a ninth aspect, the present invention provides a composition for use in medicine, wherein the composition comprises:

[0088] (i) a population of cells of the endothelial lineage that have previously been isolated according to the method of the fourth aspect; or

[0089] (ii) population of cells of the endothelial lineage and marine invertebrate collagen.

[0090] In a tenth aspect, the present invention provides a composition for use in tissue regeneration in a subject, wherein the composition comprises:

[0091] (i) a population of cells of the endothelial lineage that have previously been isolated according to the method of fourth aspect; or

[0092] (ii) a population of cells of the endothelial lineage and marine invertebrate collagen.

[0093] In an eleventh aspect, the present invention provides a method of promoting tissue regeneration in a subject comprising administering to the subject a composition comprising:

[0094] (i) a population of cells of the endothelial lineage that have previously been isolated according to the method of the fourth aspect; or

[0095] (ii) a population of cells of the endothelial lineage and marine invertebrate collagen.

[0096] In a twelfth aspect, the present invention provides a pharmaceutical composition comprising MPCs and marine invertebrate collagen. In a thirteenth aspect, the present invention provides a pharmaceutical composition comprising cells of the endothelial lineage and marine invertebrate collagen.

[0097] In some embodiments of the sixth, seventh, eighth, ninth, tenth, and eleventh aspects, the subject is a mammal. In some embodiments, the mammal is a human, mouse, rat, dog, cat, cow, sheep, goat, horse, or pig. Preferably, the subject is a human.

[0098] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects the population of MPCs or population of cells of the endothelial lineage are mammalian cells. In some embodiments, the mammalian cells are human cells, mouse cells, rat cells, dog cells, cat cells, cow cells, sheep cells, goat cells, horse cells, or pig cells. Preferably, the mammalian cells are human cells.

[0099] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is in its atelo form. In other embodiments, the marine invertebrate collagen is in its telo form.

[0100] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is thiolated.

[0101] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is cross-linked. In other embodiments, the marine invertebrate collagen is not cross-linked.

[0102] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is methacrylated.

[0103] In preferred embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is jellyfish collagen.

[0104] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the jellyfish collagen is from jellyfish of the class Scyphozoa. In some embodiments, the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof. In preferred embodiments, the jellyfish is Rhizostomas pulmo.

[0105] In some embodiments of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth aspects, the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0106] In some embodiments of the twelfth and thirteenth aspects, the composition further comprises at least one pharmaceutically acceptable compound. In some embodiments, the at least one acceptable compound is one or more selected from the group comprising: a growth factor, an antimicrobial, a cell signalling protein antagonist, an antibiotic, or an anti-inflammatory agent.

[0107] By "growth factor" we include the meaning of a naturally occurring substance capable of stimulating cellular growth, proliferation, healing, and / or cellular differentiation.

[0108] In some embodiments, the pharmaceutically acceptable compound is at least one growth factor. In some embodiments, the at least growth factor is Platelet Rich Plasma (PRP), Epithelial Growth Factor 38 (EGF), Endothelial Cell Growth Media (EGM-2), Transforming Growth Factor-Beta (TGF-B, TGF-B2, TGF-B3), Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), Granulocyte-Monocyte Colony Stimulating Growth Factor, Platelet Derived Growth Factor, Insulin-like Growth Factor 1 (IGF1), basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF), heparin, and / or ascorbic acid, or any combination thereof.

[0109] By "antimicrobial" we include the meaning of an agent that can kill or prevent the growth of microorganisms. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, "antibiotics" are used against bacteria and "antifungals" are used against fungi.

[0110] In some embodiments, the pharmaceutically acceptable compound is at least one antimicrobial. In some embodiments, the at least one antimicrobial is nano silver, penicillin, ofloxacin, tetracycline, aminoglycosides and erythromycin, flucioxacillin, clarithromycin, doxycycline, gentamicin, metronidazole, co-amoxiclav, co-trimoxazole (in penicillin), ceftriaxone, piperacillin with tazobactam, clindamycin, ciprofloxacin, vancomycin, teicoplanin, linezolid, and / or the standard of care antimicrobial agent., or any combination thereof.

[0111] By "cell signalling protein antagonist" we include the meaning of a compound that inhibits or dampens a biological response by binding to and blocking a receptor involved in a particular cell signalling cascade.

[0112] In some embodiments, pharmaceutically acceptable compound is at least one cell signalling protein antagonist. In some embodiments, the at least one cell signalling protein antagonist is calmidazolium chloride.

[0113] An "anti-inflammatory agent" is a substance that has the property of reducing inflammation and / or swelling. Examples of anti-inflammatory agents include steroidal anti-inflammatory agents (e.g. corticosteroids) and non-steroidal anti-inflammatory drugs (i.e. NSAIDs).

[0114] In some embodiments, the pharmaceutically acceptable compound is at least one antiinflammatory agent. In some embodiments, the at least one anti-inflammatory agent may be a steroidal anti-inflammatory agent such as a corticosteroid, or a non-steroidal antiinflammatory drug (NSAID) such as aspirin salsalate, diflunisal, ibuprofen, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, or sulindac.

[0115] In some embodiments of the twelfth and thirteenth aspects, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient and / or carrier. Examples of suitable pharmaceutically acceptable excipients and carriers may include sterile water, olive oil, ethyl oleate, glycols, monosaccharides such as fructose, glucose and galactose; non-reducing disaccharides such as sucrose, lactose and trehalose; non-reducing oligosacchairdes such as raffinose and melezitose; non-reducing starch derived polysaccharide products such as maltodextrins, dextrans and cyclodextrins; and non-reducing alditols such as mannitol and xylitol. Further suitable excipients include Polyethylene glycol (PEG), cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, and / or polyvinylpyrrolidone.

[0116] In a fourteenth aspect, the present invention provides a cell based medicinal product (CBMP), wherein the CBMP comprises a population of MPCs isolated, cultured, or scaffolded according to the method of the first aspect or second aspect. In a fifteenth aspect, the present invention provides a cell based medicinal product (CBMP) comprising a population of MPCs and marine invertebrate collagen.

[0117] In a sixteenth aspect, the present invention provides a method of preparing a cell based medicinal product (CBMP) according to the fourteenth aspect or the fifteenth aspect, wherein the method comprises seeding the CBMP with:

[0118] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of the first aspect or the second aspect; or

[0119] (ii) a composition comprising MPCs and marine invertebrate collagen.

[0120] By "cell based medicinal product (CBMP)" we include the meaning of medical product comprising a population of autologous and / or allogeneic cells and a device or support. The support may be in the form of a scaffold (e.g. a collagen scaffold), flowable micronised matrix, hydrogel, or mesh.

[0121] In a seventeenth aspect, the present invention provides a cell based medicinal product (CBMP), wherein the CBMP comprises a population of cells of the endothelial lineage isolated according to the method of the fourth aspect.

[0122] In an eighteenth aspect, the present invention provides a cell based medicinal product (CBMP) comprising a population of cells of the endothelial lineage and marine invertebrate collagen.

[0123] In a nineteenth aspect, the present invention provides a method of preparing a cell based medicinal product (CBMP) according to the seventeenth aspect or the eighteenth aspect, wherein the method comprises seeding the CBMP with:

[0124] (i) a population of cells of the endothelial lineage that have previously been isolated according to the method of the fourth aspect; or

[0125] (ii) a composition comprising cells of the endothelial lineage and marine invertebrate collagen.

[0126] In a twentieth aspect, the present invention provides a kit comprising: (i) a population of MPCs;

[0127] (ii) marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen; and,

[0128] (iii) instructions for use.

[0129] In a twenty-first aspect, the present invention provides a kit comprising:

[0130] (i) a population of cells of the endothelial lineage;

[0131] (ii) marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen; and,

[0132] (iii) instructions for use.

[0133] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is in its atelo form. In other embodiments, the marine invertebrate collagen is in its telo form.

[0134] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is thiolated.

[0135] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is crosslinked. In other embodiments, the marine invertebrate collagen is not cross-linked.

[0136] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is methacrylated.

[0137] In preferred embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is jellyfish collagen.

[0138] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the jellyfish collagen is from jellyfish of the class Scyphozoa. In some embodiments, the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof. In preferred embodiments, the jellyfish is Rhizostomas pulmo.

[0139] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0140] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first aspects, the population of MPCs or the population of cells of the endothelial lineage are mammalian cells. In some embodiments, the mammalian cells are human cells, mouse cells, rat cells, dog cells, cat cells, cow cells, sheep cells, goat cells, horse cells, or pig cells. Preferably, the mammalian cells are human cells.

[0141] In some embodiments of the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth aspects, the CBMP is selected from: a medical device, an implant, an injectable, a wound care product, a sponge, a scaffold, or material for use in the same.

[0142] DESCRIPTION OF THE FIGURES

[0143] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures, in which:

[0144] Figure 1 shows a comparison of the performance of PureCol® and jellyfish collagen (JFC) in maintaining sub-cultured MPCs. The graph shows metabolic activity after six days of culture of the cells, reported as a percentage of Alamar Blue reduction (%ABred) with respect to the BSA control (shown as a dotted line on the graph). The morphologies of cells cultured with BSA (control), PureCol® (bovine collagen), and JFC are shown in representative microscopy images beneath the corresponding data points in the graph.

[0145] Figure 2 shows a comparison of the performance of PureCol® and jellyfish collagen (JFC) when used in place of fibronectin during MPC endothelial early differentiation. The graph shows the number of endothelial cells produced following one week of endothelial induction in cultures with BSA (control), PureCol® (bovine collagen), and JFC. The morphologies of cells cultured with BSA (control), PureCol® (bovine collagen), and JFC following endothelial induction with EGM-2 are shown in representative microscopy images beneath the corresponding data points in the graph (upper panels). Further representative microscopy images show the results of capillary-like tube (CLT) formation assays carried out on cells harvested from each of the three culture conditions (lower panels).

[0146] Figure 3 shows a comparison of the size of calcified areas observed in MPCs over-cultured with PureCol® or jellyfish collagen (JFC). The graph shows the cumulative area of calcified deposits observed after four weeks of culture with BSA (control), PureCol® (bovine collagen), and JFC. Representative images of the size and frequency of calcified deposits (dark stained regions) observed in each culture condition are shown beneath the corresponding data points in the graph.

[0147] Figure 4 shows cellular activity of two MPC populations (P29 and P30) in 2D and 3D cultures in the presence of jellyfish collagen (JFC). 2D and 3D cultures were seeded with 0, 50,000, 100,000, or 200,000 cells. Cellular activity was reported as an increase in metabolic activity reported as percentage of Alamar Blue reduction (%ABred). Seeding efficiency (R) was determined by comparing 2D and 3D cultures seeded with the same number of cells. (A) Presents the results obtained when the AB reduction assay was carried after 2 hours incubation with the reagent. (B) Presents the results obtained when the AB reduction assay was carried after 4 hours incubation with the reagent.

[0148] Figure 5 shows the level of cell proliferation of two MPC populations (P29 and P30) 3D cultures in the presence of jellyfish collagen (JFC) and MPC-maintaining medium or MSC differentiating medium. Proliferation was reported as an increase in metabolic activity reported as percentage of Alamar Blue reduction (%ABred). (A) Presents the results obtained for P29 cells after 12 days. (B) Presents the results obtained for P30 cells after 12 days.

[0149] Figure 6 shows images from histological analysis of paraffin-embedded sections of jellyfish collagen (JFC) sponges seeded with MPCs and grown in MPC-maintaining medium. The upper panel shows a representative image at low magnification and the lower panels show regions (A) and (B) of the same image at higher magnification. The arrows in the lower panels indicate where cells are interacting with the JFC scaffold. Figure 7 shows Russell-Movat's pentachrome stain of subcutaneous implants of JFC / MPC construct cultured in StemMACS medium. (A) Presents a representative low magnification image of a section taken from a sponge seeded with MPCs and induced toward the mesengenic cell lineage using StemMACS medium. (B) Presents higher magnification images of regions indicated by the boxes in Figure 7A. The asterisks indicate cutting fractures generated during preparation of the slides and the circles indicate residual JFC.

[0150] Figure 8 shows Russell-Movat's pentachrome stain of subcutaneous implants of JFC / MPC construct cultured in ChondroDiff medium. (A) Presents a representative low magnification image of a section taken from a sponge seeded with MPCs and induced toward the chondrogenic cell lineage using ChondroDiff medium. (B) Presents higher magnification images of regions indicated by the boxes in Figure 8A. The asterisks indicate newly-formed matrix, the circles indicate residual JFC, and the arrows indicate newly-formed blood vessels.

[0151] Figure 9 shows the macroscopic appearance of JFC / MPC constructs after culture under different conditions. JFC sponges were set up no cells (control), MPCs in DMEM / 10%PhABS medium (maintaining medium), MPCs in StemMACS (mesengenic differentiation medium), and MPCs in ChondroDiff medium (chondrogenic differentiation medium). (A) Presents macroscopic images of JFC sponges after four weeks of culture. (B) Presents macroscopic images of JFC sponges after six weeks of culture.

[0152] Figure 10 shows microscopic examination of JFC / MPCs constructs cultured for four weeks under different conditions. JFC sponges were set up as follows: no cells (control); MPCs in DMEM / 10%PhABS medium (maintaining medium); MPCs in StemMACS (mesengenic differentiation medium); and, MPCs in ChondroDiff medium (chondrogenic differentiation medium). The dotted line in the DMEM / 10%PhABS (100X) image indicates where areas of collagen digestion appear to have formed. The dotted line in ChondroDiff medium (40X) image indicates an optically dense area in the scaffold.

[0153] Figure 11 shows microscopic examination of JFC sponge / MPCs constructs cultured for four weeks under chondrogenic conditions and then for a further two weeks under conditions stimulating hypertrophic terminal differentiation. (A) Russell-Movat's pentachrome stain reveals a darker area of more intense staining (area within the dotted oval) indicating the presence of newly formed cartilage. (B) Under polarised light, the non-fibrous collagen typically found in cartilage, appear dark (area within the dotted oval). (C) Safranin staining confirmed the cartilage deposition, revealing a darker area of more intense staining in that region (area within the dotted oval). (D) At higher magnification, the more intensely stained area from panel (C) resembles the typical histo-morphology of articular cartilage.

[0154] Figure 12 shows microscopic examination of JFC sponge / MPCs constructs cultured for four weeks under osteogenic differentiating condition. (A) Russell-Movat's pentachrome stain indicates that the construct is highly remodelled at the periphery, with dense fibrous collagen matrix, and in a compact central area having a different alignment of collagen fibres (area indicated by dotted line). (B) Under polarised light, the collagen at the periphery is characterised by the presence of Type I collagen (characterised by yellow-red refractivity - indicated by the arrows around the periphery of the section), while in the central area is characterised by the presence the presence of Type III collagen (characterised by greenish refractivity - indicated by the arrows towards the centre of the section).

[0155] Figure 13 shows microscopic examination of JFC sponge / MPCs constructs cultured for four weeks with a gradient of chondrogenic / osteogenic differentiating conditions across the contructs. (A) Russell-Movat's pentachrome stain indicates the formation of new organised, cartilage-like tissue on the side of the construct exposed to a higher concentration of chondrogenic differentiating media (characterised by a purple-stained area - indicated by the arrows in that panel). Conversely, on the opposite side, which was exposed to higher level of osteogenic differentiating condition, a dense matrix deposition characterised by a fibrous collagen network that stained bright pink was visible (as indicated by the asterisks in that panel). A transition area from the cartilage-like tissue to a more compact osteoid is also evident. The gradient of chondrogenic / osteogenic differentiating conditions is indicated by the arrows and triangles either side of the image. (B) and (C) Safranin staining confirmed the cartilaginous nature of the non-fibrous area, which presented as a darker area of more intense staining in that region (indicated by the arrows in those panels).

[0156] Figure 14 shows MPCs sprouting from the JFC sponges after seven days of culture under angiogenesis-stimulating condition (i.e. in the presence of EGM-2 medium). (A) Culturing of the MPC / JFC construct placed on the surface of an extracellular matrix (ECM) gel. (B) Culturing of the MPC / JFC construct embedded into an ECM gel. Results in each experimental condition show that a number of cells exhibit a "tip-cell-like" morphology, whereby the cells "invade" the ECM gel (indicated by the arrows in Panels (A) and (B)). The present invention is now further described with reference to the below examples and studies.

[0157] EXAMPLES

[0158] Example 1 - Comparison of jellyfish collagen and bovine collagen coating in MPC maintenance and differentiation

[0159] Human adult bone marrow (hBM) has been described as the elective source of Mesangiogenic Progenitor Cells (MPCs). These cells were first identified in 2009 in hBM mononuclear cells (hBM-MNCs) cultures, performed in autologous sera in place of standard FBS supplemented media, thanks to their resistance to trypsin digestion (Petrini et al, 2009. Stem Cells Dev., 18: 857-866). MPC cultures with a high grade of purity (>95%) are easily achievable by applying selective culture conditions that include medium supplementation with 10% of pooled human AB-type serum (PhABS) and use of no gas- treated hydrophobic plastic culture surface (Trombi eta / , 2009. Stem Cells Dev., 18: 1227- 1234; Montali et al, 2016. J. Vis. Exp. e54225). MPCs have been demonstrated to efficiently differentiate into exponentially growing mesenchymal stromal cells (MSCs), by activating the Wnt5 / calmodulin signaling pathway (Fazzi et al, 2011. PLoS One, 6: e25600). From the first observation, it was demonstrated that MPCs are also able to differentiate toward endothelial lineage, suggesting the term "mesangiogenic". More recently, the genuine angiogenic potential of MPCs has been assayed both in vitro and in vivo (Montali et al, 2017. Stem Cell Res. Then., 8: 106). In that work, it was clearly demonstrated that the mesengenic potential and angiogenic potential are mutually exclusive, thereby showing that MPCs lose their angiogenic potential once induced toward MSCs. MPCs have also been shown to possess longer telomeres and express pluripotency- associated markers. Oct-4, Nanog, and, in particular, nestin has been considered a marker for bone marrow-derived MPCs (Pacini et al, 2010. PLoS One, 5: e9861). Due to their unique features and differentiation potential, MPCs have been hypothesised as a new tool in developing cell-based medicinal products (CBMPs). In view of this, evaluating extracellular matrix proteins which comply with regulatory requirements for clinical application is of interest in the design of MPC-based CBMPs. Jellyfish-extracted collagen (JFC) produced by Jellagen Ltd was tested during MPC isolation, maintenance and tissue differentiation. JFC coating resulted in a higher percentage of coisolated MSCs in primary culture with respect to MPC selective culture conditions. However, further sub-culturing of the purified MPCs on JFC coated plates resulted in protection from spontaneous mesengenic differentiation, maintaining the MPCs in their undifferentiated state in DMEM / 10%PhABS. This aspect is very important, as it has been reported that the mesengenic differentiation leads to the loss of the angiogenic potential of MPCs (Montali et al, 2017. Stem Cell Res. Ther., 8: 106). Studies also confirmed that sub-culturing MPCs on JFC preserves their angiogenic potential and sprouting capability. Nonetheless, JFC coating could still support and promote induced MPC mesengenic differentiation, leading to a consistent increase in the recovery of MSCs at the end of the culture. Taken together, these results indicate JFC as being a promising natural protein matrix to apply in the culturing and delivery of MPC-based CBMPs. In order to further investigate the JFC as a medium for culturing MSCs, the culture performance of JFC and type I bovine collagen were compared in respect to in vitro applications.

[0160] The inventors investigated PureCol® (bovine collagen) produced by Advanced BioMatrix versus JFC in relation to: i) maintaining sub-cultured MPCs; ii) limiting spontaneous osteogenic differentiation; and, iii) promoting and supporting MPC angiogenic differentiation.

[0161] Main objectives:

[0162] 1) Compare the effects of JFC or PureCol® coating on maintaining MPC potential after subculturing.

[0163] 2) Compare the effects of JFC or PureCol® coating during endothelial differentiation MPC.

[0164] 3) Evaluate the induction of spontaneous osteogenic differentiation during over-culturing in mesengenic medium on JFC or PureCol® coating. Methods

[0165] Cell culture

[0166] MPCs were isolated using standard selective culture conditions: DMEM / 10%PhABS on hydrophobic plastics for six days and detached by TypLE Select. 20,000 cells / cm2were seeded in 6-well plates with different treatment of culture surface: BSA only, JFC, or PureCol®. Three plates per sample were prepared and cultured with:

[0167] (A) Mantaining medium-. DMEM / 10%PhABS;

[0168] (B) Angiogenic medium-. EGM-2 (Lonza); or

[0169] (C) Mesengenic differentiating medium-. StemMACS MSC expansion medium (Miltenyi Biotec).

[0170] (A) Cultures were maintained for six days before AlamarBlue (AB) reduction assays were performed.

[0171] (B) Cultures were maintained for one week and then cells were enzymatically digested. 50,000 cells detached from BSA, JFC, or PureCol® cultures were seeded on Geltrex® thick gels in order to perform capillary-like tube formation assay (CLT assay) in EGM-2 medium.

[0172] (C) Cells were over-cultured* for four weeks, then fixed and stained with Alizarin S. Alizarin S is a stain used to identify calcium-containing osteocytes derived from differentiated MSCs.

[0173] *Over-cultured cells = cells that have cultured to confluence and then stressed in order to induce spontaneous ossicle formation.

[0174] Coating

[0175] 6-well plates were coated with 12pg / cm2collagen by applying 0.2mL / cm2of the coating solution for 2-4 hours. Coating Solution (ImL):

[0176] - ImL of D-PBS;

[0177] - lOpL of Bovine Serum Albumin (BSA) (lOOpg / mL); and

[0178] - 20pL Collagen solution*

[0179] ^Collagen solution used was either:

[0180] - Jellyfish Collagen solution (3mg / ml); or

[0181] - PureCol® (3mg / ml).

[0182] The coating solution was removed, and the plates immediately used for cell culture. For each plate, two wells were coated with JFC, two wells with PureCol® and two others with 0.2mL / cm2of Ipg / mL BSA as control wells.

[0183] Results

[0184] Objective 1

[0185] The results obtained confirm previous data suggesting that JFC coating protects subcultured MPCs from spontaneous mesengenic differentiation into proliferating MSCs. Indeed, both morphology and metabolic activity of cells cultured on JFC (reported as percentage of Alamar Blue reduction (%ABred) with respect to the control) was comparable to the BSA control (112.6 ±2.8%, n = 5). In contrast, subculturing MPCs on PureCol®, after six days of culture in DMEM / 10%PhABS, resulted in the loss of MPC morphology and a 20% reduction in the metabolic activity of the cells (79.2 ±4.9%, p<0.001, n = 5) (Figure 1).

[0186] Objective 2

[0187] It was observed that sub-culturing MPCs on JFC not only protects from uncontrolled mesengenic differentiation, but also preserves their angiogenic potential. No differences in their ability to sprout from 3D-speroids under VEGF stimulus was observed. The sprouting from spheroids represents a first step of MPC endothelial differentiation (Montali et al, 2017. Stem Cell Res. Then, 8: 106). However, this early differentiation could also be achieved in 2D cultures, usually on plastics coated with fibronectin (Fazzi et al, 2011. PLoS One, 6: e25600). In these experiments, 2D early endothelial differentiation of MPCs was performed, substituting fibronectin with the coatings described above in Objective 1 (i.e. BSA only, JFC, or PureCol®). Interestingly, culturing under inducing conditions on JFC seems to significantly promote endothelial differentiation. After one week, twice the number of endothelial cells were harvested from JFC endothelial induction than from BSA control cultures (9.35 ±1.08 xlO5vs 5.11 ±1.13xl05, p<0.05, n = 5). In contrast, performing endothelial induction on PureCol® lead to a lower number of cells when compared to the BSA control, (Figure 2 upper panel). To verify that the harvested cells could be considered genuine pre-endothelial cells, a capillary-like tube (CLT) formation assay was performed. Cells obtained from all of the three surface coatings were CLT-positive, however, the capillary-like tubes formed from cells on BSA showed significantly smaller lumen areas and segment lengths, when compared to the two collagen-based experiments (Figure 2 lower panel).

[0188] Objective 3

[0189] During long-term over-culturing, cells were able to grow in multi-layers, without signs of the cells suffering, independently from the surface coatings. However, after four weeks, and even in the absence of external osteogenic stimulus, sporadic deposition of calcified matrix was observed. Moreover, the number and size of calcium deposits (dark spots in Figure 3 lower panel), as evaluated by quantification of Alizarin S-positive area, was significantly increased in PureCol® cultures (24.26 ±6.56 xl03 pm2, n=4, p<0.05) when compared to either the BSA control (6.11 ±1.78 xl03 pm2, n=4) or JFC (7.42 ±1.66 xl03 pm2, n=4). Alizarin S-positive was quantified on calibrated microphotographs covering the entire culture surface for each test well. The values were derived by image analysis as a percentage of dark pixels in the total pixel area.

[0190] Conclusions

[0191] The results in this Example confirm previous data demonstrating the preservation of MPC differentiating potential when sub-culturing with JFC coated surfaces. Furthermore, the present results demonstrate that JFC possesses a superior culture performance during MPC angiogenic induction, in particular in promoting differentiation and supporting proliferation of pre-endothelial cells. In contrast, the use of bovine collagen has a negative effect on MPC maintenance and in fact promotes spontaneous, and uncontrolled osteogenic differentiation.

[0192] These results also provide evidence of an in vitro supporting / promoting activity of JFC on early angiogenesis. This observation is also significant in relation to medical applications of MPCs, as new blood vessel formation is of particular importance in the early phase of tissue regeneration, when new-formed tissue should be sufficiently vascularised to support the cell proliferation and differentiation required.

[0193] Example 2 - Culture of MPCs on jellyfish collagen sponges

[0194] Experiment 1 - MPCs seeded on J FC sponges

[0195] Materials and methods

[0196] Two samples of MPCs (Samples P29 and P30) were collected from a primary culture of bone marrow (BM) aspirates under standard selective culture conditions (see Example 1 above).

[0197] 3D Jellagen® JFC sponges (sized for 96-well plates) were loaded with 50,000, 100,000 or 200,000 freshly isolated MPCs. Sponges without cells were also used as controls. In order to determine seeding efficiency, 2D cultures were also set up with equivalent numbers of seeded cells. All experiments were performed in duplicate. Following 24 hours incubation, an AlamarBlue (AB) reduction assay was performed after incubation for 2 hours or 4 hours with the reagent.

[0198] Results

[0199] JFC Scaffolds alone led to a mild reduction of AB (i.e. lower than 10%). A linear positive correlation was detected between %ABred and increasing cell seeding densities, for both P29 and P30 samples. Seeding efficiency (R) was observed to be from about 60% to 70%, and was independent of the number of cells loaded (Figures 4A and 4B). Experiment 2 -MPCs cultured in MPC maintaining medium or MSC differentiating medium

[0200] Materials and methods

[0201] Sponges from Experiment 1 above were then transferred to a new plate and cultured for 12 days in MPC maintaining medium (DMEM / 10%PhABS) or in MSC differentiating medium (StemMACS MSC Exp). AB reduction assays were then repeated to evaluate the cell proliferation. Constructs were then fixed, and paraffin embedded for histological analysis.

[0202] Results

[0203] A significant increase in cell activity was detected in maintaining medium (see Figures 5A and 5B) after twelve days, with respect to the 24 hour assay (dotted lines), with apparently higher proliferation rate at 50,000 and 100,000 initial cell density, in both samples. The P29 sample showed a consistent increase of proliferation in differentiating media, independent of the initial cell density, whereas P30 showed a plateau as cell density increased. Histological analysis was carried out on the sponges seeded with MPCs and grown in MPC-maintaining medium (Figure 6, upper panel) and revealed a number of cells interacting with the scaffold (Figure 6, lower panel), as indicated by the arrows.

[0204] Conclusions

[0205] It is possible to load from 50,000 to 200,000 MPCs per scaffold without loss of cells due to saturation of the scaffolds, and with a seeding efficiency around 60-70%.

[0206] Example 3 - Implantation of jellyfish collagen sponges loaded with cultured MPCs in a mouse model

[0207] MPCs were collected from primary culture of BM aspirates under standard selective culture Conditions (see Example 1). Two 3D Jellagen® JFC sponges (size for 96-well) were loaded with 300,000 freshly isolated MPCs and constructs were cultured for 12 days in StemMACS or ChondroDiff media, as partial induction respectively, toward mesengenic or chondrogenic lineage. Following culturing, constructs were transplanted into the backs of nude mice in a surgically-produced sub-cutaneous pocket. The pockets were sutured and mice were sacrificed six weeks later. Implants were then fixed and processed for histological examination through application of haematoxylin and eosin stain (H / E) and Russell-Movat's pentachrome stain.

[0208] Results

[0209] At the macroscopic level, heterotopic implants were easily found and excised during mice autopsy. No signs of inflammation and / or fibrosis in the surrounding tissues were detected.

[0210] Notably, once embedded in paraffin, the implants induced towards mesengenic lineage (StemMACS: 12 days culture before surgery) were difficult to cut into 5 pm slices for histological analysis.

[0211] At the microscopic level, Russell-Movat's pentachrome stain revealed that the implant induced towards the mesengenic lineage (dashed line in Figure 7A) contained homogenously compact, non-vascularised, but highly cellularised tissue. When compared to the surrounding tissues, the implant was characterised by several fractures, which are attributable to the cutting procedure (asterisk in Figure 7B). The scaffold had the appearance of being completely resorbed except for very rare residual JFC fibres (circle in Figure 7B). The newly-formed tissue present appears to be predominantly Type I / II collagen and osteoid matrix, respectively presenting as bright red and yellow regions following staining with Russell-Movat's penthorome stain (as indicated by the arrows in Figure 7B). Violet stain also revealed that cartilaginous matrix is homogenously dispersed. Taken together, these results indicate the production of newly-formed tissue that is highly comparable to the tissues of primary ossification described in bone development and repair.

[0212] In contrast to the above, the pre-transplantation 12-day culture in ChondroDiff media (i.e. induced toward a chondrogenic lineage) resulted in a completely different histology in the implant (Figure 8A). Specifically, the implant appeared much less compact and cellularised and there was limited scaffold resorbing, resulting in an almost conserved JFC structure (as shown by the circles in Figure 8B). However, several scaffold pores appeared "filled" or partially filled by newly-formed tissue (as shown by asterisks in Figure 8B). This is coloured in very pale violet over a yellowish background. Notably, the implant was highly vascularised by perfused blood vessels (as shown by the arrowheads in Figure 8B, with red blood cells present), in particular at the periphery of the implant. Taken together, these results suggest that under these experimental conditions the cell proliferation within the implant could be inhibited in favour of increased angiogenic processes. Moreover, new matrix deposition seems incomplete without signs of scaffold remodelling.

[0213] Conclusions

[0214] In summary, JFC sponges represent an advantageous scaffold for use in MPC transplantation, and support the proliferation and differentiation of MPCs in vivo. Furthermore, pre-conditioning of JFC / MPC constructs affects the behaviour of MPCs in vivo. For instance, it appears that in vitro mesengenic induction may trigger cell proliferation in the early phase, followed by stimulated osteogenic differentiation that leads to the formation of non-vascularised calcified ossicles. Conversely, pre-induction toward the chondrogenic fate results in an apparent impairment of cell proliferation accompanied by an increased vascularisation of the implants. However, initial cell dose of 300,000 cells / scaffold seems insufficient for the deposition and maturation of compact cartilaginous tissue. It would therefore be of interest evaluate JFC / MPC constructs at higher cell seeding doses.

[0215] Example 4 - Macroscopic and microscopic evaluation of jellyfish collagen soonae and MPC constructs at higher cell doses after culturing

[0216] Materials and methods

[0217] Samples of MPCs were collected from a primary culture of BM aspirates under standard selective culture conditions (see Example 1 above).

[0218] Samples PP37, AN2, AN9, BS190121, and MF070920 exhibited very high recovery of MPCs (from 3.5 to 5.0 million of cells). Thus, it was possible to test JFC loading with high cell doses. Four re-hydrated JFC sponges (size for 96-well) were seeded with 1.5xl06freshly isolated MPCs and allowed to adhere overnight in DMEM / 10%PhABS. On the following day, media was changed and constructs were cultured in three different conditions:

[0219] (A) DMEM / 10%PhABS MPC (maintaining);

[0220] (B) StemMACS MSCs Expansion medium MPC (mesengenic differentiation);

[0221] (C) ChondroDiff medium MPC (direct chondrogenic differentiation);

[0222] (D) OsteoDiff medium MPC (direct osteogenic differentiation);

[0223] (E) ChondroDiff / OsteoDiff gradient MPC (direct osteo-chondral differentiation);

[0224] (F) Sprouting angiogenesis (direct vasculogenic differentiation)

[0225] The number of PP37 cells obtained was not sufficient for loading three sponges. Accordingly, only conditions (B) and (C) tested for PP37 cells, and the cells were maintained for six weeks.

[0226] In relation to AN2 cells, sponges for each of conditions (A), (B), and (C) were prepared, and the cells were maintained for four weeks. In relation to AN9 and MF070920 cells, sponges for each condition (B), (C), and (D) were prepared, and the cells were maintained for two, four, or six weeks. In relation to BS190121 cells, sponges for each condition (D) and (E) were prepared, and the cells were maintained for two or four weeks.

[0227] A re-hydrated JFC sponge without any cells loaded was also maintained in condition (B) as a negative "no cells" control.

[0228] After culturing, constructs were washed in D-PBS and fixed in 4% Paraformaldehyde for 1 hour. After extensive washing, samples were dehydrated through application of 50% and 70% ethanol and maintained in 70% ethanol until processing for histological analysis.

[0229] Results

[0230] At the macroscopic level, no differences were observed between the JFC / MPC constructs cultured in DMEM / 10%PhABS (i.e. condition (A)) or StemMACS (i.e. condition (B)) and the "no cells" control after four weeks of culture. In contrast, the JFC / MPCs construct cultured in ChondroDiff medium (i.e. condition (C)) seemed to have contracted with an approximate 50% reduction in size, and much more compact appearance when compared to the control and conditions (A) and (B). Indeed, the chondrogenic induction resulted in a construct with higher mechanical properties that was resistant to compression. Most of the construct volume seems constituted by brilliant white and translucent compact matrix very similar to hyaline cartilage (Figure 9A).

[0231] After six weeks of culture, the chondrogenic induction (i.e. condition (C)) resulted in the complete loss of the discoidal shape of the construct. Instead, the construct took on a compact spheroid appearance (approximately 50% smaller than the "no cells" control), and homogeneously constituted hyaline cartilage-like tissue. After six weeks of culture, the JFC / MPCs construct maintained in StemMACS (the MSCs differentiating / expanding medium) (i.e. condition (B)) showed an approximately 30% size reduction and more compact appearance. However, it had inferior mechanical properties (i.e. a jelly-like consistency) when compared to chondrogenic differentiated constructs (i.e. condition (C)) (Figure 9B).

[0232] The six-week cultured constructs could not be microscopically inspected due to high matrix condensation preventing light transmission. However, it was possible to analyse the four- week cultured JFC / MPCs constructs under low-power magnification (Figure 10). Under 40X and 100X magnification, the "no cells" control maintained its spongy structure, presenting an uncorrupted and complex web of cross-linked collagen fibres. A similar scaffold structure was also detected after culturing JFC / MPCs constructs in StemMACS medium (condition (B)). However, a consistent number of small and rounded cells were homogenously dispersed in the collagen sponge cultured under condition (B). In contrast, JFC / MPCs constructs maintained in DMEM / 10%PhABS (i.e. condition (A)) showed a mildly remodelled collagen scaffold in which some areas of collagen digestion appeared to have formed (area within the dotted line in Figure 10, first row of panels).

[0233] The JFC / MPC constructs cultured in ChondroDiff medium (i.e. condition (C)) showed a large and optically dense area (dark area encircled by dotted lines) in Figure 10 which is indicative of new tissue formation. Histological analysis using Russell-Movat's pentachrome and Safranin stains characterised that newly-formed tissue as being cartilage-like tissue (Figures 11A, 11C, and 11D). Further confirmation of the presence of cartilage-like tissue was obtained from analysis of the Russell-Movat's pentachrome stained samples when exposed to polarised light. Under those conditions, the areas of the samples with non-fibrous collagen (i.e. in hyaline cartilage) appeared darker because no refractivity was observed, which is characteristic of the presence of hyaline cartilage (Figure 11B). The JFC / MPC constructs cultured in OsteoDiff medium (i.e. condition (D)) showed a optically dense peripheral region and a large, compact central area following staining with Russel I- M ova t's pentachrome stain (compact central area indicated within the dotted line in Figure 12A). Under polarised light, the peripheral regions of the Russell-Movat's pentachrome stained samples exhibits a distinctive yellow-red refractivity, which is normally associated with the presence of Type I collagen (arrows pointing to bright areas at the outside edge of the sample in Figure 12B). In contrast, the central and compact areas of the resulted in a characteristic green refractivity, which is typical of Type III collagen (arrows pointing to bright areas toward the centre and upper centre of the sample in Figure 12B).

[0234] After four weeks of culture under ChondroDiff / OsteoDiff gradient differentiating conditions (i.e. condition (E)), JFC / MPC constructs appeared highly remodelled with a large area resembling articular cartilage stained purple (arrows in the image in Figure 13A) facing the chondrogenic stimulus (represented by the hatched arrows either side of Figure 13A) and an optically dense and compact area stained pink indicating the presence of fibrous collagen facing the osteogenic stimulus (represented by the black arrow). Those two different newly formed tissues appeared to be interconnected, with a "transition zone" having formed between them. Safranin staining confirmed the cartilaginous nature of the non-fibrous area, which presented as a darker area of more intense staining, indicated by the arrow into that region (Figures 13B and 13C).

[0235] In addition, MPCs were demonstrated to be able to sprout under angiogenesis-stimulating conditions (i.e. condition (F)) when scaffolded onto JFC sponges at high cell number (i.e. 300,000 to 5 million cells) (Figures 14A and 14B).

[0236] Conclusions

[0237] High cell dose constructs cultured in DMEM / 10%PhABS (i.e. condition (A)) maintain MPCs in their undifferentiated state with signs of local collagen scaffold degradation, whereas mesengenic stimulus (i.e. condition (B)) support proliferation of MSC-like cells without signs of scaffold remodelling or matrix deposition. Moreover, the results indicate that a cell dose of 1.5 million cells per scaffold supports the formation of hyaline cartilage from JFC / MPC constructs under in vitro chondrogenic stimulus (i.e. conditions (C) and (E)). When in vitro osteogenic stimulus is applied (i.e. conditions (D) and (E)), production of compact and fibrous collagen-containing tissue (osteoid-like) was observed. Taken together, these results show that MPCs can rapidly and specifically respond to both chondrogenic and ostegenic differentiation stimuli when cultured in vitro in combination with JFC sponges.

[0238] Embodiments of the invention will now be described in the following numbered paragraphs:

[0239] (1). A method of selectively isolating a population of mesangiogenic progenitor cells

[0240] (MPCs), wherein the method comprises culturing a bone marrow-derived sample in the presence of marine invertebrate collagen and a culture medium.

[0241] (2). A method of culturing or scaffolding a population of mesangiogenic progenitor cells

[0242] (MPCs), wherein the method comprises culturing or scaffolding MPCs in the presence of marine invertebrate collagen and a culture medium.

[0243] (3). The method according to paragraph 2, wherein the method further comprises isolating the population of MPCs.

[0244] (4). The method according to any one of paragraphs 1 to 3, wherein the marine invertebrate collagen is in its atelo form.

[0245] (5). The method according to any one of paragraphs 1 to 3, wherein the marine invertebrate collagen is in its telo form.

[0246] (6). The method according to any one of paragraphs 1 to 5, wherein the marine invertebrate collagen is thiolated.

[0247] (7). The method according to any one of paragraphs 1 to 6, wherein the marine invertebrate collagen is cross-linked.

[0248] (8). The method according to any one of paragraphs 1 to 7, wherein the marine invertebrate collagen is methacrylated.

[0249] (9). The method according to any one of paragraphs 1 to 8, wherein the marine invertebrate collagen is jellyfish collagen.

[0250] (10). The method according to paragraph 9, wherein the source of the jellyfish collagen is from jellyfish of the class Scyphozoa.

[0251] (11). The method according to paragraph 10, wherein the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof.

[0252] (12). The method according to paragraph 11, wherein the jellyfish is Rhizostomas pulmo.

[0253] (13). The method according to any one of paragraphs 1 to 12, wherein the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0254] (14). The method according to any one of paragraphs 1 to 13, wherein the marine invertebrate collagen is coated onto a tissue culture surface.

[0255] (15). The method according to paragraph 14, wherein the marine invertebrate collagen is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

[0256] (16). The method according to any one of paragraphs 1 to 15, wherein the method prevents or reduces differentiation of the population of MPCs into cells of the mesenchymal stromal cell (MSC) lineage.

[0257] (17). The method according to paragraph 16, wherein the population of MPCs comprises less than 20% of cells of the MSC lineage, preferably less than 15% of cells of the MSC lineage, more preferably less than 10% of cells of the MSC lineage, most preferably less than 5% of cells of the MSC lineage.

[0258] (18). The method according to any one of paragraphs 1 to 17, wherein the culture medium is MPC-maintaining medium.

[0259] (19). The method according to paragraph 18, wherein the MPC-maintaining medium comprises Dulbecco's Modified Eagles Medium (DMEM) and serum.

[0260] (20). The method according to any one of paragraphs 1 to 19, wherein the method promotes maintenance of the population of MPCs in their undifferentiated state. (21). The method according to any one of paragraphs 1 to 20, wherein the population of

[0261] MPCs have MPC differentiation potential.

[0262] (22). The method according to paragraph 20 or 21, wherein the population of MPCs have

[0263] MPC vasculogenic differentiation potential.

[0264] (23). The method according to paragraph 20 or 21, wherein the population of MPCs are capable of differentiating into cells of the endothelial cell lineage.

[0265] (24). The method according to any one of paragraphs 20 to 23, wherein the MPCs are capable of differentiating into blood vessel cells, cartilage cells, muscle, and / or bone cells.

[0266] (25). The method according to paragraphs 20 to 24, wherein after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential and / or are capable of differentiating into cells of the endothelial cell lineage is 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%, at least 95%, preferably at least 70%, more preferably 80%, yet more preferably 90%, most preferably 95%.

[0267] (26). The method according to any one of paragraphs 1 to 25, wherein the population of

[0268] MPCs are mammalian cells.

[0269] (27). The method according to paragraph 26, wherein the mammalian cells are human cells.

[0270] (28). A population of MPCs obtained according to the method of any one of paragraphs 1 to 27.

[0271] (29). A method of obtaining a population of cells of the endothelial lineage, wherein the method comprises culturing a population of MPCs in the presence of marine invertebrate collagen and a culture medium that promotes differentiation of the population of MPCs into cells of the endothelial lineage. (30). The method according to paragraph 29, wherein the cells of the endothelial cell lineage comprise blood vessel-forming cells and / or endothelial cells.

[0272] (31). The method according to paragraph 29 or paragraph 30, wherein the culture medium comprises Endothelial Cell Growth Media (EGM-2) and serum.

[0273] (32). The method according any one of paragraphs 29 to 31, wherein the marine invertebrate collagen is in its atelo form.

[0274] (33). The method according any one of paragraphs 29 to 31, wherein the marine invertebrate collagen is in its telo form.

[0275] (34). The method according to any one of paragraphs 29 to 33, wherein the marine invertebrate collagen is thiolated.

[0276] (35). The method according to any one of paragraphs 29 to 34, wherein the marine invertebrate collagen is cross-linked.

[0277] (36). The method according to any one of paragraphs 29 to 35, wherein the marine invertebrate collagen is methacrylated.

[0278] (37). The method according to any one of paragraphs 29 to 36, wherein the marine invertebrate collagen is from jellyfish.

[0279] (38). The method according to paragraph 37, wherein the source of the jellyfish collagen is from the class Scyphozoa.

[0280] (39). The method according to paragraph 37 or paragraph 38, wherein the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., or any combination thereof.

[0281] (40). The method according to paragraph 39, wherein the jellyfish is Rhizostomas pulmo. (41). The method according to any one of paragraphs 29 to 40, wherein the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

[0282] (42). The method according to any one of paragraphs 29 to 41, wherein the marine invertebrate collagen is coated onto a tissue culture surface.

[0283] (43). The method according to paragraph 42, wherein the marine invertebrate collagen is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

[0284] (44). The method according to any one of paragraphs 29 to 43, wherein the population of MPCs are mammalian cells.

[0285] (45). The method according to paragraph 44, wherein the mammalian cells are human cells.

[0286] (46). A population of cells of the endothelial lineage obtained according to the method of any one of paragraphs 29 to 45.

[0287] (47). A composition for use in medicine, wherein the composition comprises:

[0288] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of paragraphs 1 to 27; or

[0289] (ii) a population of MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

[0290] (48). A composition for use in tissue regeneration in a subject, wherein the composition comprises:

[0291] (i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of paragraphs 1 to 27; or

[0292] (ii) a population of MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen. (49). The composition for use according to paragraph 47 or paragraph 48, wherein the subject is a mammal, optionally wherein the mammal is a human.

[0293] (50). A pharmaceutical composition comprising MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

[0294] (51). The pharmaceutical composition according to paragraph 50, wherein the composition further comprises at least one pharmaceutically acceptable compound.

[0295] (52). A cell based medicinal product (CBMP) comprising a population of MPCs isolated, cultured, or scaffolded according to the method of any one of paragraphs 1 to 27.

[0296] (53). A cell based medicinal product (CBMP) comprising a population of MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

[0297] (54). A cell based medicinal product (CBMP) according to paragraph 52 or paragraph 53, wherein the CBMP is selected from: a medical device, an implant, a wound care product, a sponge, a scaffold, or material for use in the same.

[0298] (55). A method of preparing a cell based medicinal product (CBMP) according to any one of paragraphs 52 to 54, comprising seeding the CBMP with:

[0299] (i) A population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of paragraphs 1 to 27; or

[0300] (ii) A composition comprising a population of MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

[0301] (56). A method of promoting tissue regeneration in a subject comprising administering to the subject a composition comprising:

[0302] (i) A population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of paragraphs 1 to 27; or

[0303] (ii) A population of MPCs and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen. (57). A kit comprising:

[0304] (i) a population of MPCs;

[0305] (ii) marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen; and,

[0306] (iii) instructions for use.

Claims

Claims1. A method of selectively isolating a population of mesangiogenic progenitor cells (MPCs), wherein the method comprises culturing a bone marrow-derived sample in the presence of marine invertebrate collagen and a culture medium.

2. A method of culturing or scaffolding a population of mesangiogenic progenitor cells (MPCs), wherein the method comprises culturing or scaffolding MPCs in the presence of marine invertebrate collagen and a culture medium.

3. The method according to claim 2, wherein the method further comprises isolating the population of MPCs.

4. The method according to any one of claims 1 to 3, wherein the marine invertebrate collagen is in its atelo form or its telo form.

5. The method according to any one of claims 1 to 4, wherein the marine invertebrate collagen is thiolated, and / or cross-linked, and / or methacrylated.

6. The method according to any one of claims 1 to 5, wherein the marine invertebrate collagen is jellyfish collagen, optionally wherein the source of the jellyfish collagen is from jellyfish of the class Scyphozoa, optionally wherein the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., . or any combination thereof.

7. The method according to any one of claims 1 to 6, wherein the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

8. The method according to any one of claims 1 to 7, wherein the marine invertebrate collagen is coated onto a tissue culture surface, optionally wherein the marine invertebrate collagen is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

9. The method according to any one of claims 1 to 8, wherein the method prevents or reduces differentiation of the population of MPCs into cells of the mesenchymal stromal cell (MSC) lineage, optionally wherein the population of MPCs comprises less than 20% of cells of the MSC lineage, preferably less than 15% of cells of the MSC lineage, more preferably less than 10% of cells of the MSC lineage, most preferably less than 5% of cells of the MSC lineage.

10. The method according to any one of claims 1 to 9, wherein the culture medium is MPC-maintaining medium, optionally wherein the MPC-maintaining medium comprises Dulbecco's Modified Eagles Medium (DMEM) and serum.

11. The method according to any one of claims 1 to 10, wherein the method promotes maintenance of the population of MPCs in their undifferentiated state.

12. The method according to any one of claims 1 to 11, wherein the population of MPCs have MPC differentiation potential.

13. The method according to claim 11 or 12, wherein the population of MPCs have MPC vasculogenic differentiation potential.

14. The method according to claim 11 or 12, wherein the population of MPCs are capable of differentiating into cells of the endothelial cell lineage.

15. The method according to any one of claims 11 to 14, wherein the cells of the endothelial cell lineage are capable of differentiating into blood vessel cells, cartilage cells, muscle, and / or bone cells.

16. The method according to any one of claims 11 to 15, wherein after at least 24 hours of culturing or scaffolding the percentage of cells of the population of MPCs that have MPC differentiation potential and / or are capable of differentiating into cells of the endothelial cell lineage is 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%, at least 95%, preferably at least 70%, more preferably 80%, yet more preferably 90%, most preferably 95%.

17. The method according to any one of claims 1 to 16, wherein the population of MPCs are mammalian cells, optionally wherein the mammalian cells are human cells.

18. A population of MPCs obtained according to the method of any one of claims 1 to 17.

19. A method of obtaining a population of cells of the endothelial lineage, wherein the method comprises culturing a population of MPCs in the presence of marine invertebrate collagen and a culture medium that promotes differentiation of the population of MPCs into cells of the endothelial lineage.

20. The method according to claim 19, wherein the cells of the endothelial cell lineage comprise blood vessel-forming cells and / or endothelial cells.

21. The method according to claim 19 or 20, wherein the culture medium comprises Endothelial Cell Growth Media (EGM-2) and serum.

22. The method according any one of claims 19 to 21, wherein the marine invertebrate collagen is in its atelo form or its telo form.

23. The method according to any one of claims 19 to 22, wherein the marine invertebrate collagen is thiolated, and / or cross-linked, and / or methacrylated.

24. The method according to any one of claims 19 to 23, wherein the marine invertebrate collagen is from jellyfish, optionally wherein the source of the jellyfish collagen is from the class Scyphozoa, optionally wherein the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Rhopilema hispidum, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Catostylus sp., Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., and Lobonema sp., . or any combination thereof.

25. The method according to any one of claims 19 to 24, wherein the marine invertebrate collagen is in the form of a coating, sponge matrix, a nano-fibre electrospun matrix, or a hydrogel.

26. The method according to any one of claims 19 to 25, wherein the marine invertebrate collagen is coated onto a tissue culture surface, optionally wherein the marine invertebrate collagen is present at a concentration of at least lpg / cm2, more preferably at least 5pg / cm2, most preferably at least 10pg / cm2.

27. The method according to any one of claims 19 to 26, wherein the population of cells of the endothelial lineage are mammalian cells, optionally wherein the mammalian cells are human cells.

28. A population of cells of the endothelial lineage obtained according to the method of any one of claims 19 to 27.

29. A composition for use in medicine, wherein the composition comprises:(i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of claims 1 to 16 or a population of cells of the endothelial lineage isolated according to the method of any one of claims 19 to 27; or(ii) a population of MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

30. A composition for use in tissue regeneration in a subject, wherein the composition comprises:(i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of claims 1 to 16 or a population of cells of the endothelial lineage isolated according to the method of any one of claims 19 to 27; or(ii) a population of MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

31. The composition for use according to claim 29 or 30, wherein the subject is a mammal, optionally wherein the mammal is a human.

32. A pharmaceutical composition comprising MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen, optionally wherein the composition further comprises at least one pharmaceutically acceptable compound.

33. A cell based medicinal product (CBMP) comprising a population of MPCs isolated, cultured, or scaffolded according to the method of any one of claims 1 to 17 or a population of cells of the endothelial lineage isolated according to the method of any one of claims 19 to 27.

34. A cell based medicinal product (CBMP) comprising a population of MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen, optionally wherein the CBMP is selected from: a medical device, an implant, a wound care product, a sponge, a scaffold, or material for use in the same.

35. A method of preparing a cell based medicinal product (CBMP) according to claim 33 or 34, comprising seeding the CBMP with:(i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of claims 1 to 17 or a population of cells of the endothelial lineage isolated according to the method of any one of claims 19 to 27; or(ii) a composition comprising a population of MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

36. A method of promoting tissue regeneration in a subject comprising administering to the subject a composition comprising:(i) a population of MPCs that have previously been isolated, cultured, or scaffolded according to the method of any one of claims 1 to 17 or a population of cells of the endothelial lineage isolated according to the method of any one of claims 19 to 27; or(ii) a population of MPCs or a population of cells of the endothelial lineage, and marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen.

37. A kit comprising:(i) a population of MPCs or a population of cells of the endothelial lineage;(ii) marine invertebrate collagen, optionally wherein the marine invertebrate collagen is jellyfish collagen; and,(iii) instructions for use.

Images