WO2022155377A1

WO2022155377A1 - Bovine umbilical cord stem cells as feeder layer for embryonic stem cell propagation

Info

Publication number: WO2022155377A1
Application number: PCT/US2022/012365
Authority: WO
Inventors: Dorit ELIYAEV
Original assignee: Meatech 3D, Ltd.; The IP Law Firm of Guy Levi, LLC
Priority date: 2021-01-13
Filing date: 2022-01-13
Publication date: 2022-07-21

Abstract

The disclosure relates to methods, and compositions for propagating bovine embryonic stem cells (bESC), on a feeder layer. Specifically, the disclosure is directed to methods and compositions for the xeno-free propagation of bESC on bovine umbilical stem cells (bUCSC), derived from a bovine umbilical cord.

Description

BOVINE UMBILICAL CORD STEM CELLS AS FEEDER LAYER FOR EMBRYONIC STEM CELL PROPAGATION

BACKGROUND

[0001] The disclosure is directed to methods, and compositions for propagating bovine embryonic stem cells (bESC), on a feeder layer. Specifically, the disclosure is directed to methods and compositions for the xeno-free propagation of bESC on bovine umbilical stem cells (bUCSC), derived from a bovine umbilical cord.

[0002] In an effort to reduce the impact of animal agriculture and to improve people's nutrition, as well as for various other incentives, there is a need for alternatives to animal meat for development of novel protein sources containing viable cells culture(s) that correspond to the three-dimensional (3D) tissue, for instance, muscle tissue.

[0003] Embryonic stem (ES) cells are special kind of cells that can both duplicate themselves (in other words, divide and proliferate indefinitely in culture) and produce differentiated functionally specialized cell types. These stem cells are capable of becoming almost all of the specialized cells of the body and thus, may have the potential to generate cells for a broad array of tissues such as, for example, muscle, cartilage and the like.

[0004] Feeder layers were applied to support the prolonged growth of pluripotent stem cells (PSCs) such as ES cells for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for SC cultures. However, these feeder layers from animal that is different from the desired differentiated PSC can increase the dissimilarities between the tissue grown and the feeder layer potentially compromising the propagation and differentiation of PSC sought to be differentiated thereon.

[0005] The following disclosure provides therefore xeno-free culture system for a more humane growth and expansion of bESC.

SUMMARY

[0006] Disclosed, in various implementations, are methods and compositions for xeno-free propagation of bESC on bovine umbilical stem cells (bUCSC) derived from a bovine umbilical cord. [0007] In an implementation provided herein is a method for xeno-free propagation of bovine embryonic stem cells (bESC), comprising culturing bESC on a feeder layer of bovine umbilical cord stem cells (bUCSC) isolated from a plurality of bovine umbilical cords.

[0008] In another implementation, isolating the MSCs from the plurality of bovine umbilical cords comprises: providing the plurality of intact bovine umbilical cords; following longitudinal dissection, removing umbilical arteries, umbilical vein, and vasa-vasorum; chopping the remaining Wharton’s Jelly; of isolating the bUCSC form the bovine umbilical cord comprises: providing a plurality of intact bovine umbilical cords; following longitudinal dissection, removing umbilical arteries, umbilical vein, and vasa vasorum; chopping the remaining Wharton’s Jelly; disaggregating a plurality of mesenchymal stem cells (MSCs) in the Wharton’s Jelly; depositing the disaggregated MSCs on a substrate as a monolayer; seeding the monolayer with a bESC composition and incubating the seeded monolayer for a predetermined period; treating said MSCs with mitomycin C to stop proliferation; seeding bESCs - post isolation - on top of UC-MSCs for culture and expansion.

BRIEF DESCRIPTION OF THE FIGURES

[0009] For a better understanding of the method for propagating xeno-free bovine embryonic stem cells (bESC), on a feeder monolayer of bovine umbilical cord stem cells (bUCSC), with regard to the implementations thereof, reference is made to the accompanying examples and figures, in which:

[00010] FIG. 1, is a cross-section schematic of a mammalian umbilical cord transverse to the umbilical cords longitudinal axis.

DETAILED DESCRIPTION

[00011] Provided herein are implementations of methods and compositions for propagating xeno-free bovine embryonic stem cells (bESC, referring to undifferentiated cells derived from the inner cell mass of bovine embryos that have the potential to become any specialized cell), comprising culturing bESC on a feeder monolayer (sheets) of bovine umbilical cord stem cells (bUCSC) isolated from a plurality of bovine umbilical cords.

[00012] Bovine ES cells (bESC) - (referring to a pluripotent cell characterized by the properties of ESC such as, for example, proliferation without transformation, infinite replication, selfrenewal and differentiation into all three germ layers; endoderm, mesoderm, and ectoderm), extracted from the ICM during the blastocyst stage, can be cultured in-vitro and, under the right conditions, can proliferate indefinitely. Bovine ESC growing in this undifferentiated state retain the potential to differentiate into all three germ layers-derived tissues (namely at least one of: an endoderm, an ectoderm, and a mesoderm).

[00013] In an exemplary implementation, a xeno-free culture system using bUCSC as a feeder layer disclosed is beneficial for growth and propagation of bESC and for further differentiation in the effort to produce desired tissues in vitro. The use of bovine umbilical cords as a cell source is desirable since it is plentiful, inexpensive, and can be obtained through noninvasive validated procedures that are repeatable and reliable. In the context of the disclosure, the term “xeno-free” means that the origin of the culture system is not from a foreign source, i.e. does not contain material of non-bovine animal origin when bovine stem cells are to be cultured on the bovine umbilical cord derived feeder layer.

[00014] In the context of the disclosure, the term “feeder layer” refers to cells used in co-culture for a desired effect, for example, to maintain pluripotent stem cells. Likewise, the term "pluripotent cell" refers to a cell that has the potential to develop into any cell type(s).

[00015] Accordingly and in an exemplary implementation, provided herein is a method for propagating xeno-free bovine embryonic stem cells (bESC), comprising culturing bESC on a feeder layer of bovine umbilical cord stem cells (bUCSC) isolated from a bovine umbilical cord.

[00016] The step of culturing is preceded by a step isolating the bUCSC from the bovine umbilical cord, whereby isolating the bUCSC form the bovine umbilical cord (UC) comprises: providing a plurality of intact bovine umbilical cords of those of cows where there were no complications in either the pregnancy, or the parturition of the calves. Following dissection of the collected UCs, along their longitudinal axis, umbilical arteries, umbilical vein, and vasa vasorum (see e.g., FIG. 1) are removed; and the remaining Wharton’s Jelly is chopped fine (e.g. to pieces having a size of between about 2 mm³ and about 5 mm³), whereupon the chopped Wharton’s Jelly is transferred to (coated) resin substrate, where the MSCs are isolated from the tissue by enzymatic disaggregation, using effective disaggregation agent. The isolated MSCs is seeded on the substrate for expansion and propagation on tissue culture dishes. Upoa predetermined number of passages, treating the MSCs with mitomycin C to stop proliferation; and seeding bESCs - post isolation - on top of UC-MSCs monolayer sheets for culture and expansion.

[00017] In the context of the disclosure, the term “disaggregating agent” means a fluid like a buffer comprising a substance used to destroy the anchorage of target cells within the tissue without influencing the target cells itself. This anchorage derives from interactions of the cells with the extracellular matrix or with adjacent cells. These interactions, e.g. tight junctions, gap junctions, desmosomes, and hemidesmosomes, are built mainly by proteins, e.g. cadherins, connexins, claudins and integrins, mostly in a calcium-dependent manner Therefore, the release agent which destroys the tissue integrity may contain a calcium-free and/or a calcium-depleting agent and/or enzymes that degrade the extracellular matrix or extracellular protein-protein interactions. The administration of the components of the release agent may be sequentially or simultaneously.

[00018] For example, the agent to disaggregate the biological tissue (6) is selected from the group consisting of trypsin, chymotrypsin, papain, collagenase, elastase, dispase, thermolysin, hyaluronidase, clostripain and neutral protease from clostridium histolyticum, pronase, DNase I, pepsin, proteinase K, lysozyme, chelating agents for bivalent ions (like EDTA or citrate) and mixtures thereof.

[00019] The seeded MSC cells are then incubated (e.g., at a temperature of between 35°C and 38 °C) for a predetermined period (e.g., between about 20 min and about 120 min.). In the context of the disclosure, the term "Wharton's jelly" refers to the gelatinous substance located on the exterior of the amnion separating the chorion and amnion, in other words, the amniotic membrane surrounding the Wharton's jelly of the umbilical cord, and not comprising thick collagen fibers and/or ducts (e.g., the Allantoic duct, see FIG.l) and vascular walls (villi and intervening chambers).

[00020] In an exemplary implementation, the step of transferring is preceded by: digesting the chopped Wharton’s Jelly with an effective amount of at least one of: collagenase, DNAase, and pronase for a predetermined period; and filtering the digested Wharton’s Jelly, thereby removing undigested tissue and tissue debris. For example, the effective amount (referring to the concentration operable to produce the desired results) can be, between about 0.02% (v/v) and about 0.08% collagenase, between about 0.0005% and about 0.0015% DNase I, and between about 0.15% and about 0.25% pronase, for a period of between about 45 min. and about 75 min at, for example, 37°C. Likewise, filtering of the digested Wharton’s Jelly can be done in another exemplary implementation, using 100-pm mesh filter, to remove undigested tissue clumps and debris.

[00021] In an exemplary implementation, the filtered UC-derived MSCs are further resuspended at a predetermined ratio in a medium composition comprising DMEM supplemented with FBS, L-glutamine, and at least one of: penicillin, and streptomycin. The resuspension medium composition for the UC-derived MSCs can be, for example a composition comprising between about 70% (v/v) and about 90% DMEM, between about 30% (v/v) and about 10% FBS, and between about 20% (v/v) and about 0.5% of at least one of: penicillin, and streptomycin, at a ratio of 80:20:1 respectively. The resuspended, digested and filtered UC-derived MSCs can then be cultured for a predetermined period of between 2 and 7 days at 37 °C under controlled atmosphere, for example, between 0.3% (v/v) and about 10% CO2.

[00022] In certain exemplary implementation, it may be desired to mitotically inactivate the monolayer UC-derived MSCs cells. In the methods disclosed, inactivation can be done mitotically and/or lethally by treating the monolayer UC-derived MSCs cells with a method effective for inhibiting or preventing unlimited proliferation of stem cells. For example, using irradiation, phototherapy, or any agent that inhibits cell proliferation including but not limited to inhibitors of cellular components necessary for mitosis such as but not limited to protein synthesis, microtubule function, spindle check point unit, cell cycle specific kinases, cyclins, and or apoptotic inducing agents, as well as any means of genetic, protein, and or cell manipulation that will allow termination or prevention of unregulated or unlimited cell proliferation. This can be done, in an exemplary implementation, by contacting the UC-derived MSCs with a composition comprising at least one of: a taxane (e.g., Cabazitaxel, Docetaxel, Nab-paclitaxel, or Paclitaxel.), an alkaloid (e.g., Vinca Alkaloids, Cholchicine), methylazoxymethanol acetate (MAM), etoposide, teniposide, ixabepilone, estramustine, eribulin, and a composition comprising two or more of the foregoing.

[00023] The UC-derived MSCs suspension medium (e.g., DMEM supplemented with FBS, L- glutamine, and at least one of: penicillin, and streptomycin) comprises in certain implementations, a predetermined amount of non-essential amino acids (e.g., at least one of: alanine, glutamine (glutamic acid), serine, asparagine (aspartic acid), glycine, and proline).

[00024] In an implementation, the step of culturing (referring to the growth of cells in vitro in an artificial medium for commercial purpose), of the bESC, can comprise culturing the bESC in the presence of (or in plating over) the feeder layer medium. For the bESC culture, feeder layers can further comprise, for example, bovine embryonic fibroblasts (BEF) that have been treated to prevent them from dividing in culture. Furthermore, the bESC medium, can be, for example, at least one of: a composition comprising: Dulbecco's modified Eagle's medium (DMEM) without Sodium pyruvate having glucose content of between about 70% and about 90%; between about 10% and about 30% Fetal bovine serum (FBS); 0 -mercaptoethanol (0.1 mM); about 1% of non-essential amino acids; L- Glutamine 2 mM; and basic fibroblast growth factor (BFGF), a composition comprising: Minimum Essential Medium Alpha (MEM-oc) with 10% inactivated fetal calf serum, and a composition comprising: DMEM; 15% Fetal bovine serum; Penicillin/ Streptomyocin; Glutamine; Non-essential amino acids; nucleosides; P-mercaptoethanol; Sodium pyruvate; and leukaemia inhibitory factor (LIF).

[00025] Other embryo culture and maturation media routinely used for the collection and maintenance of embryos, used in connection with the methods disclosed, can be, for example, Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Eactate-Pyruvate (TAEP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media. For example TCM-199, and 1 to 20% serum supplement including fetal calf serum, newborn calf serum, estrual cow serum, or steer serum. An example of maintenance medium can be TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mM pyruvate and 50 pg/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of bovine cell types forming a feeder layer such as, at least one of: granulosa cells, oviduct cells, BRE cells, and uterine cells. Furthermore, the step of culturing the bESC further comprises, in certain implementations, mechanically dissociating (e.g., using a pipette) the cultured bESC (colonies) and re-plating the bESC on substrates (e.g., petri dishes) coated with as-yet unseeded feeder layer composition comprising bUCSC disclosed herein.

[00026] The bESC may be subsequently cultured in differentiation medium, operable to cause the culture to differentiate to at least one of: a muscle cell, a cartilage cell, a fat cell, and a connective tissue cell. The term “differentiate” and its derivatives, as used with respect to cells in a differentiating cell system, refers to the process by which cells differentiate from one cell type (e.g., a multipotent, totipotent or pluripotent differentiable cell) to another cell type such as a target-differentiated cell (e.g., a muscle cell, a cartilage cell, a fat cell, a neuron cell, and the like). For example, differentiation into hematopoietic and endothelial cell lineages in the presence of (exposed to) a composition comprising: bFGF; stem cell factor; and oncostatin. M. Myocytes differentiation can be done by, for example, by employing pharmacologic inhibitors and agonists (e.g., for upregulation of Mesogeninl) as well as isolated cytokines or other protein-based signals, for example, to cause overexpression of myogenic transcription factor MyoD. In other words, a process whereby the unspecialized bESC acquires the features of a specialized cell such as a muscle cell. Differentiation is controlled by the interaction of the cells’ genes with the physical and chemical conditions outside the cell, for example, through signaling pathways involving proteins embedded in the cell surface.

[00027] In the methods for the xeno-free propagation of bESC on bovine umbilical stem cells (bUCSC) described herein, the step of seeding the bESC is preceded by forming a suspended mixture of the bESC. The suspended cells can be, for example, cells’ dispersion, solution, emulsion, suspension, hydrogel or liquid compositions. Furthermore, culturing the isolated bESC further comprises mechanically dissociating the bESC and re-plating the bESC on the feeder layer disclosed.

[00028] Under certain circumstances, the compositions used in the methods disclosed, can further comprise other additives that affect colonization, proliferation, adherence, inhibit apoptosis or other manipulation of the cells, retain native growth of the cells, and/or organ and/or tissue and other similar functions, for example: cells manipulation triggering compounds, (for example, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), insulin-like growth factor (IGF), glucoseaminoglycan (GAG), Transforming growth factor (TGF) or signaling compound composition comprising the foregoing), as well as therapeutically effective compounds, antimicrobial compounds, immunosuppressing compounds and the like.

[00029] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[00030] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. “Combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms “a”, “an” and “the” herein do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The suffix “(s)” as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the cell(s) includes one or more cell). Reference throughout the specification to “one implementation”, “another implementation”, “an implementation”, “an exemplary implementation” and so forth, when present, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the implementation is included in at least one implementation described herein, and may or may not be present in other implementations. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various implementations.

[00031] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Furthermore, the term "about" means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is "about" or "approximate" whether or not expressly stated to be such. For example, “about” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[00032] Accordingly and in an exemplary implementation, provided herein is a method for propagating bovine embryonic stem cells (bESC), comprising culturing bESC on a feeder layer of bovine umbilical cord stem cells (bUCSC) isolated from a bovine umbilical cord, wherein (i) the step of culturing is preceded by a step isolating the bUCSC from the bovine umbilical cord, (ii) the step of isolating the bUCSC form the bovine umbilical cord comprises: providing a plurality of intact bovine umbilical cords; following longitudinal dissection, removing umbilical arteries, umbilical vein, and vasa vasorum; chopping the remaining Wharton’s Jelly; disaggregating a plurality of mesenchymal stem cells in the Wharton’s Jelly; depositing the disaggregated cells on a substrate as a monolayer seeding the monolayer with a bESC composition and incubating the seeded monolayer for a predetermined period, wherein (iii) the step of transferring is preceded by: digesting the chopped Wharton’s Jelly with an effective amount of at least one of: collagenase, DNAase, and pronase for a predetermined period; and filtering the digested Wharton’s Jelly, thereby removing undigested tissue and tissue debris, the method further comprising (iv) resuspending the filtered cells at a predetermined ratio in a medium composition comprising DMEM supplemented with FBS, L-glutamine, and at least one of: penicillin, and streptomycin; and culturing the resuspended cells, and (v) periodically resuspending the cultured cells at a predetermined ratio within the medium composition, wherein (vi) the step of culturing comprises culturing the resuspended cells in a modified atmosphere, wherein (vii) the medium composition further comprises a predetermined amount of non-essential amino acids, (viii) the step of culturing the bESC further comprises mechanically dissociating the bESC and re-plating the bESC on the feeder layer, wherein (ix) the embryonic stem cell medium is a composition comprising: “Knockout DMEM” without Sodium pyruvate having glucose content of between about 70% and about 90%; between about 10% and about 30%knockout serum replacment; □- mercaptoethanol (0.1 mM); about 1% of non-essential amino acids; L-Glutamine 2 mM; and basic fibroblast growth factor (BFGF), wherein (x) the step of culturing the bESC is carried out in the presence of a bESC medium, wherein (xi) the bESC medium is at least one of: a composition comprising: Dulbecco's modified Eagle's medium (DMEM) without Sodium pyruvate having glucose content of between about 70% and about 90%; between about 10% and about 30% Fetal bovine serum (FBS); 0 -mercaptoethanol (0.1 mM); about 1% of non-essential amino acids; L- Glutamine 2 mM; and basic fibroblast growth factor (BFGF), Minimum Essential Medium Alpha (MEM- > ) with 10% inactivated fetal calf serum, and a composition comprising: Dulbecco's modified Eagle's medium (DMEM); 15% Fetal bovine serum; Penicillin/ Streptomyocin; Glutamine; Non- essential amino acids; nucleosides; 0-mercaptoethanol; Sodium pyruvate; and leukaemia inhibitory factor (LIF), (xii) the step of culturing in the presence of the bESC medium, is followed by a step admixing a differentiation medium, wherein (xiii) the differentiation medium is operable to cause the culture to differentiate to at least one of: a muscle cell, a cartilage cell, a fat cell, and a connective tissue cell, the method further comprising (xiv) coating the substrate with a biocompatible hydrogel comprising gelatin, further comprising (xv) mitotically inactivating the monolayer UC-derived MSCs, by (xvi) contacting the substrate with a composition comprising at least one of: a taxane, an alkaloid, methylazoxymethanol acetate (MAM), etoposide, teniposide, ixabepilone, estramustine, eribulin, and a composition comprising two or more of the foregoing.

[00033] Although the foregoing disclosure for xeno-free propagation of bESC on bovine umbilical stem cells (bUCSC) has been described in terms of some implementations, other implementations will be apparent to those of ordinary skill in the art from the disclosure herein. Moreover, the described implementations have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel methods, systems and compositions described herein may be embodied in a variety of other forms without departing from the spirit thereof. Accordingly, other combinations, omissions, substitutions and modifications will be apparent to the skilled artisan in view of the disclosure herein.

Claims

What is claimed:

1. A method for propagating bovine embryonic stem cells (bESC), comprising culturing bESC on a feeder layer of bovine umbilical cord stem cells (bUCSC) isolated from a bovine umbilical cord.

2. The method of claim 1, wherein the step of culturing is preceded by a step isolating the bUCSC from the bovine umbilical cord.

3. The method of claim 2, wherein the step of isolating the bUCSC form the bovine umbilical cord comprises: a) providing a plurality of intact bovine umbilical cords; b) following longitudinal dissection, removing umbilical arteries, umbilical vein, and vasa vasorum; c) chopping the remaining Wharton’s Jelly; d) disaggregating a plurality of mesenchymal stem cells in the Wharton’s Jelly; e) depositing the disaggregated cells on a substrate as a monolayer f) seeding the monolayer with a bESC composition and g) incubating the seeded monolayer for a predetermined period.

4. The method of claim 3, wherein the step of transferring is preceded by: a) digesting the chopped Wharton’s Jelly with an effective amount of at least one of: collagenase, DNAase, and pronase for a predetermined period; and b) filtering the digested Wharton’s Jelly, thereby removing undigested tissue and tissue debris.

5. The method of claim 4, further comprising: a) resuspending the filtered cellsat a predetermined ratio in a medium composition comprising DMEM supplemented with FBS, L-glutamine, and at least one of: penicillin, and streptomycin; and b) culturing the resuspended cells.

6. The method of claim 5, further comprising periodically resuspending the cultured cells at a predetermined ratio within the medium composition.

7. The method of claim 5, wherein the step of culturing comprises culturing the resuspended cells in a modified atmosphere.

8. The method of claim 5, wherein the medium composition further comprises a predetermined amount of non-essential amino acids.

9. The method of claim 1, wherein the step of culturing the bESC further comprises mechanically dissociating the bESC and re-plating the bESC on the feeder layer.

10. The method of claim 6, wherein the embryonic stem cell medium is a composition comprising: a) “Knockout DMEM” without Sodium pyruvate having glucose content of between about 70% and about 90%; b) between about 10% and about 30%knockout serum replacment; c) ^mercaptoethanol (0.1 mM); d) about 1% of non-essential amino acids; e) L- Glutamine 2 mM; and f) basic fibroblast growth factor (BFGF).

11. The method of claim 10, wherein the step of culturing the bESC is carried out in the presence of a bESC medium.

12. The method of claim 11, wherein the bESC medium is at least one of: a) a composition comprising: i. Dulbecco's modified Eagle's medium (DMEM) without Sodium pyruvate having glucose content of between about 70% and about 90%; ii. between about 10% and about 30% Fetal bovine serum (FBS); iii. P -mercaptoethanol (0.1 mM); iv. about 1% of non-essential amino acids; v. L- Glutamine 2 mM; and vi. basic fibroblast growth factor (BFGF), b) Minimum Essential Medium Alpha (MEM-oc) with 10% inactivated fetal calf serum, and c) a composition comprising: i. Dulbecco's modified Eagle's medium (DMEM); ii. 15% Fetal bovine serum; iii. Penicillin/ Streptomyocin; iv. Glutamine; v. Non-essential amino acids; vi. nucleosides; vii. P-mercaptoethanol; viii. Sodium pyruvate; and ix. leukaemia inhibitory factor (LIF).

13. The method of claim 12, wherein the step of culturing in the presence of the bESC medium, is followed by a step admixing a differentiation medium.

14. The method of claim 13, wherein the differentiation medium is operable to cause the culture to differentiate to at least one of: a muscle cell, a cartilage cell, a fat cell, and a connective tissue cell.

15. The method of claim 3, further comprising coating the substrate with a biocompatible hydrogel comprising gelatin.

16. The method of claim 5, further comprising mitotically inactivating the monolayer UC-derived MSCs.

17. The method of claim 16, wherein the step of mitotically inactivating the monolayer UC- derived MSCs, comprises contacting the substrate with a composition comprising at least one of: a taxane, an alkaloid, methylazoxymethanol acetate (MAM), etoposide, teniposide, ixabepilone, estramustine, eribulin, and a composition comprising two or more of the foregoing.