Oct4, a transcription factor belonging to the fifth class of POU proteins (POUV), plays essential roles in the maintenance of pluripotency, differentiation and the generation of induced pluripotent stem cells (iPSCs). Oct4 regulates two levels of pluripotency, which are distinguished by their gene expression profiles and epigenetic status, namely the naïve and primed state of pluripotency. Embryonic stem cells (ESCs) and embryonic germ cells (EGCs), which are isolated from inner cell mass and primordial germ cells in the embryo, respectively, are in vitro models in which the naïve state is propagated through self-renewal. Epiblast stem cells (EpiSCs) and traditional human ESCs have gene expression profiles that are closest to the post-implantation epiblast, which is closer to embryonic differentiation, and exhibit a primed state of pluripotency. As Oct4 is important for pluripotency in all these cell types, where it regulates different targets, it appears to have two distinct sets of functions, namely germ cell/naïve ESC-like activity and epiblast/primed pluripotency-like activity. Based on protein sequences and syntenic gene analysis, Oct4/POUV homologs of jawed vertebrates can be classified into two subfamilies: POU5F1 and POU5F3, which are thought to originate from a genome duplication event that occurred in a common ancestor. Most extant vertebrates have lost one of these paralogs, while a small fraction, including coelacanths, axolotls, turtles, and marsupials, retains both POUV forms. In my thesis, I investigated the gene duplication event that underlies divergence of POU5F1 and POU5F3 in both expression pattern and specialised function. In particular, I focused on species that have retained both genes and asked whether POUV functional divergence correlates with ancestral origin. To test the function of POU5F1 and POU5F3, I substituted endogenous mouse Oct4/Pou5f1 with different POUV proteins using a cell line in which endogenous Oct4 expression can be silenced with tetracycline (ZHBTc4). Results showed that POU5F1 proteins had a greater capacity to support naïve ESC pluripotency and self-renewal than POU5F3 proteins. Global transcriptome analysis of the POUV-rescued ESC lines revealed that coelacanth POU5F1 protein regulates gene expression in a similar manner to mouse Oct4, in that genes involved in stem cell maintenance, reproduction and development are upregulated in ESCs rescued by POU5F1, but not POU5F3. Coelacanth POU5F3 rescued lines, however, expressed genes involved in various cell differentiation programs, including cell adhesion (e.g. E-cadherin and N-cadherin). This suggests that POU5F3 plays a role in primed pluripotency, while POU5F1 regulates naïve pluripotency. However, there is one POU5F3 factor that rescues ESCs like Oct4, the Xenopus gene Xlpou91 (Pou5f3.1). In Xenopus, a further duplication of POU5F3 gene enabled specialization, and Xlpou91 is expressed specifically in the primordial germ cells. Xlpou25 (Pou5f3.2) exhibits epiblast-specific activities and lacks the capacity to maintain naïve ESC pluripotency, similar to other POU5F3 proteins. This functional distinction between the different Xenopus POUV paralogs enabled us to address how specific Oct4 functions (germ cell-like versus epiblast-like activity) are related to the induction of pluripotency. To address this question, mouse Oct4 was replaced by either Xlpou91 or Xlpou25 in murine cellular reprogramming using a Nanog-GFP reporter line to monitor iPSC generation. Results showed that Xlpou91 and mouse Oct4 were required at similar levels to reprogram somatic cells toward iPSCs and reprogrammed cells emerged with similar kinetics. Conversely, Xlpou25 was required at higher expression levels and the resulting iPSCs appeared at a later timepoint, while the pluripotent population in these cultures appeared to be less stable and more prone to differentiate. I found that this phenotype of enhanced differentiation in Xlpou25 reprogrammed cultures may be a product of a different set of immediate early genes induced at the first stages of differentiation. Global transcriptome analysis of the naïve ESC-like pluripotent subpopulation of these iPSC lines confirmed the capacity of all Xenopus POUVs to drive reprogramming towards the pluripotent state. However, the gene sets induced by both Xlpou91 and mouse Oct4, but not Xlpou25, were somewhat enriched for genes involved in reproduction, emphasizing the segregated role of Xlpou91 as a germ cell specific POUV protein. Lastly, I explored the evolutionary origin of these two POUV paralogs and attempted to identify a POUV-related gene in jawless vertebrate (cyclostomes). Based on in silico analysis of genomic and transcriptome databases, my collaborators and I were able to identify a single POUV gene in the Japanese/arctic lamprey, thus providing the first insight into the origin of gnathosome POUV genes.

No description available.

L’absence de traitements efficaces pour régénérer l’encéphale a orienté les recherches vers un nouveau domaine : la thérapie cellulaire. Dans ce contexte, les cellules souches apparaissent comme un outil de choix pour reformer le tissu cérébral et restaurer les capacités cognitives et les cellules souches olfactives ecto-mésenchymateuses (CSOE-Ms), localisées dans la cavité nasale, apparaissent comme un candidat idéal. Chez des rats, nous avons démontré que la transplantation de CSOE-Ms restaurait les capacités mnésiques impactées après une ischémie et entraînait une augmentation de la neurogenèse dans l’hippocampe.Nous avons ensuite promu l’intérêt de ces cellules auprès de la communauté scientifique et du monde vétérinaire en caractérisant les cellules de huit espèces d’intérêt. Ces travaux ont révélé de fortes similitudes entre les cellules de toutes les espèces, ce qui permet de généraliser en partie les propriétés thérapeutiques de ces cellules depuis les données obtenues majoritairement à partir de cellules humaines. Afin de confirmer ces observations in vitro, nous avons effectué des greffes dans le quatrième ventricule cérébral de vieux chiens présentant un syndrome dégénératif lié au vieillissement. L’injection de cellules souches a permis de restaurer en partie les capacités cognitives perturbées et s’est révélé sans aucun effet délétère.Les résultats obtenus au cours de cette thèse confirment l’intérêt des CSOE-Ms pour traiter les atteintes du système nerveux central et suggèrent qu’elles pourraient être un outil majeur pour la médecine régénérative. / The lack of effective treatments to regenerate the brain led research into a new field: the cellular therapy. In this context, stem cells appear as a tool of choice to regenerate brain tissues and restore cognitive abilities. Olfactory ecto-mesenchymal stem cells (OE-MSCs), located in the nasal cavity, appear as an ideal candidate.In rats, we have demonstrated that transplantation of OE-MSCs restored memory capacities affected after ischemia and led to an increase in neurogenesis within the hippocampus.We then promoted the interest of these cells toward the scientific and veterinary community by characterizing the cells of eight species of interest. This work revealed strong similarities between the cells of all the species, which makes it possible to generalize in part their therapeutic properties from the data obtained mainly from human cells.To confirm these observations in vitro, we performed transplants in the fourth cerebral ventricle of old dogs with degenerative syndrome related to aging. The injection of stem cells partially restored disturbed cognitive abilities and was shown to have no deleterious effect.The results obtained during this thesis confirm the interest of OE-MSCs to treat central nervous system injuries and suggest that they could be a major tool for regenerative medicine.

Self-renewing, chemoresistant cancer cells that contribute to cancer metastasis and patient relapse have properties similar to those of stem cells, and have been termed "cancer stem cells" (CSCs) in the literature. The identification of signaling pathways that regulate CSC development and/or function is an important step towards understanding why patients relapse, and towards development of novel therapeutics that specifically target CSC vulnerabilities. Recent studies have identified a role for the aryl hydrocarbon receptor (AHR), an environmental carcinogen receptor implicated in cancer initiation, in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in CSC development. To test this hypothesis, AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells was modulated with AHR ligands, shRNA, or AHR-specific inhibitors and their phenotypic, genomic, and functional CSC characteristics were evaluated. Aldehyde dehydrogenase (ALDH) was used as an epithelial stem cell marker for flow cytometry. Results demonstrate that: 1) ALDHhigh cells express elevated levels of Ahr and the AHR-driven gene that encodes cytochrome p450 isoform 1b1 (Cyp1b1), 2) AHR knockdown reduces ALDH activity, 3) AHR hyper-activation significantly increases ALDH1 activity, expression of stem cell- and invasion/migration-associated genes, and accelerates cell migration, 4) a highly significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of the CSC- and invasion/migration-associated gene sets was seen with genomic data obtained from 79 human breast cancer cell lines and over 1850 primary human breast cancers, 5) the AHR interacts directly with the transcription factors Sox2 and Runx1, and AHR ligands increase this interaction, 6) AHR knockdown inhibits tumorsphere formation in low adherence conditions, 7) AHR inhibition blocks the rapid migration of ALDHhigh cells and reduces ALDHhigh cell chemoresistance, and 8) AHR knockdown inhibits tumor growth and reduces tumor Aldh1a1, Sox2, and Cyp1b1 expression in orthotopic xenografts. These data suggest that the AHR plays an important role in development of CSCs in a large fraction of human breast cancers and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of CSC-like properties.

Elucidating the molecular events that integrate the patterning, morphogenesis, and differentiation of epithelial progenitor cells into complex tissues is a primary focus of epithelial developmental biology research. Expansion and maintenance of epithelial progenitor populations is crucial for developmental events, but growth must be tightly coupled to consequent cellular differentiation and specialization. The Hippo pathway has surfaced as an important regulator of epithelial progenitor identity: nuclear activity of the Hippo effector Yap maintains epithelial progenitor status while Hippo-mediated nuclear exclusion of Yap by the Lats1/2 kinases induces differentiation. Extending this general theme into an additional organ system, the submandibular gland (SMG), as well as identifying upstream regulators of Yap and Lats1/2 in the developing lung was the goal of this work. Here, we describe important roles for Yap in the morphogenesis and patterning of lung and SMG epithelium, both of which are composed of highly organized branched structures. Epithelial-specific genetic ablation of Yap as well as its upstream negative regulators Lats1/2 was used to interrogate loss- and gain-of-function phenotypes, whereby Lats1/2 ablation is known to result in unrestricted nuclear Yap activity. Loss of Yap in the SMG resulted in a striking deficiency of Krt5/Krt14-positive epithelial progenitor populations accompanied by impaired branching morphogenesis. Deletion of Lats1/2 in the SMG resulted in a massive expansion of Krt5/Krt14-positive epithelial progenitor populations that failed to terminally differentiate. As epithelial progenitors in the lung and SMG begin to differentiate, they also acquire distinct morphologies. In both the lung and the SMG, Krt5-positive basal cells lie beneath a layer of Krt8/Krt19-positive luminal cells. We observed that luminal cells exhibited a columnar morphology while basal cells retained a cuboidal morphology, and that this difference correlated with the expression of the polarity protein Crb3. After ablating Crb3 in the developing lung epithelium, luminal cells were unable to polarize, exhibited aberrant nuclear Yap activity, and remained in a progenitor state. Crb3 functions to initiate Lats1/2 activity, promoting Yap phosphorylation and its consequent nuclear exclusion, which drives differentiation. Taken together, this work identifies essential roles for polarity/Hippo pathway-mediated control of Yap activity in epithelial progenitor expansion and differentiation. / 2018-07-09T00:00:00Z

Successful mammalian development to term requires that embryonic and extra-embryonic tissues communicate and grow in coordination, to form the body. After implanting into the uterus, the mouse embryo is comprised of three cell lineages: first, the embryonic epiblast (EPI) that forms the embryo proper, second, the extra-embryonic ectoderm (ExE) which contributes to the foetal portion of the placenta, and third, the visceral endoderm (VE) that contributes to the yolk sac. These three tissues form a characteristic ‘egg-cylinder’ structure, which allows signals to be exchanged between them and sets the stage for body axis establishment and subsequent tissue patterning. The mechanisms underlying this process are difficult to study in vivo because a different genetically manipulated mouse line must be generated to investigate each factor involved. This difficulty has prompted efforts to model mammalian embryogenesis in vitro, using cell lines, which are more amenable to genetic manipulation. The pluripotent state of the EPI can be captured in vitro as mammalian embryonic stem cells (ESCs). Although mouse ESCs have been shown to contribute to all adult tissues in chimeric embryos, they cannot undertake embryogenesis when allowed to differentiate in culture. Previous studies have shown that ESCs formed into three-dimensional (3D) aggregates, called embryoid bodies, can become patterned and express genes associated with early tissue differentiation. However, embryoid bodies cannot recapitulate embryonic architecture and therefore may not accurately reflect what happens in the embryo. In this study, a new technique was developed to model early mouse development which is more faithful to the embryo. ESCs were co-cultured with stem cells derived from the ExE, termed trophoblast stem cells (TSCs), embedded within extracellular matrix (ECM). These culture conditions lead to the self-assembly of embryo-like structures with similar architecture to the mouse egg cylinder. They were comprised of an embryonic compartment derived from ESCs abutting an extra-embryonic compartment derived from TSCs, and hence were named ‘ETS-embryos’. These structures developed a continuous cavity at their centre, which formed via a similar sequence of events to those that lead to pro-amniotic cavity formation in the mouse embryo, and required active Nodal/Activin signalling. After cavitation, ‘ETS-embryos’ developed regionalised mesodermal tissue and primordial germ cell-like cells originating at the boundary between embryonic and extra-embryonic compartments. Inhibitor studies revealed that this occurred in response to endogenous Wnt and BMP signalling, pathways which also govern these tissue specification events in the early mouse embryo. To demonstrate that ‘ETS-embryos’ were comparable to mouse embryos at the global transcriptional level, RNA-sequencing was then performed on different tissue regions of ‘ETS-embryos’ and the resulting transcriptomes were compared to datasets from mouse embryos. These data showed that ‘ETS-embryos’ were highly similar to mouse embryos at post-implantation stages in their overall gene expression patterns. Taken together, these results indicate that ‘ETS-embryos’ are an accurate in vitro model of mammalian embryogenesis, which can be used to complement studies undertaken in vivo to investigate early development.

Human pluripotent stem cell (hPSC)-derived neuronal and glial material presents a relatively new opportunity to model human neurophysiology in both health, and disease. Validation of regionally-defined hPSC-derived neurones and glia cultures thus represents the founding blocks of technology that aims to complement existing models. Principally, the relevance of in vitro hPSC-derived material is determined by how representative it is of native material, yet at present the physiology of these cells remains underexplored. Here, electrophysiology and pharmacology are used to functionally assess hPSC-derived excitatory cortical neurones (hECNs), motorneurones (MNs) and oligodendrocyte-lineage cells in the context of regional-specific properties and maturation. These properties are then examined in material derived from hPSCs generated from patients with neurological disorders. This thesis examines of the properties of GABAARs and strychnine-sensitive glycine receptors (GlyRs) in hECNs by assessing their subunit composition, and compares these with studies which have made comparable investigations of rodent tissue where maturation is associated with a shift in GABAA and GlyR compositions. Using pharmacology and RNAseq analysis, GABAAR and GlyRs in hECNs were found to possess receptor populations typical of those reported in the immature cortex. hECNs generated from patients harbouring a mutation to the Disrupted-in-schizophrenia-gene 1 (DISC1), a candidate schizophrenia gene, were then examined. Imbalances in the excitation/inhibition balance are suspected in schizophrenia and, in this regard, the intrinsic excitability properties alongside expression and composition of major neurotransmitter receptors and intracellular chloride concentration were assessed. No obvious differences in excitability or functional expression of AMPARs, GABAARs or NMDARs were observed between case and control derived neurones. Receptor composition and intracellular chloride concentrations were found to be predominantly immature-like, however, AMPAR composition and intracellular chloride concentration were found to be like that of adult cortical neurones. These data are discussed in the context of modelling DISC1-associated pathologies. Thirdly, MNs from hPSCs generated from ALS patients harbouring mutations on the C9ORF72 gene were examined. The hypothesis that increased glutamate-mediated excitoxicity could, in part, be explained by increased expression of Ca2+- permeable AMPARs was examined. The estimated mean single-channel conductance of AMPARs was found to be high in MNs derived from ALS patients, reminiscent of Ca2+-permeable AMPARs and was reversed by gene-editing of the C9ORF72 mutation. Finally, oligodendrocytes generated from ALS patients harbouring TARDBP mutations were examined. Distinctive electrophysiological shifts in oligodendrocytes-lineage cell development are reported. A similar AMPAR phenotype of elevated Ca2+-permeable AMPAR expression was observed in oligodendrocytes derived from two patient hPSC lines and was rescued in an isogenic, gene-edited line, raising the intriguing possibility of convergence in pathophysiologies in the nature of the overlap between cell-type, AMPAR pathology and excitotoxicity in ALS disease progression mechanisms.

Paediatric intestinal failure following bowel resection causes significant morbidity and mortality. There is a pressing need for improved treatment modalities. Following loss of bowel, the remaining intestine undergoes a period of adaptation, characterised by an increase in height of the intestinal villi. Better understanding the factors that govern the formation and growth of villi may lead to therapeutic interventions that amplify the intrinsic adaptation response. This thesis aims to explore the processes by which intestinal villi form during embryological development, the contribution of intestinal stem cells to this, and candidate signalling pathways that may yield insights into new therapeutic interventions for patients with intestinal failure. Abstract: Aim I will examine the morphogenesis and morphology of intestinal villi by investigating three themes: 1) Villus morphogenesis: When and where do villi form along the gut tube? Can this process be quantified, both in vivo and in vitro? Is this initiated by a dominolike signaling-cascade along the bowel, or location-specific intrinsic triggers? 2) Stem cells: What is the spatiotemporal appearance of the Lgr5-expressing intestinal stem cells during development? How does this relate to the process of villus morphogenesis? 3) Signalling pathways: Can a genetic mutation mouse model help elucidate pathways by which post bowel resection adaptation might occur? Can this be used to help identify potential intestinotrophic agents? Abstract: Materials and Methods Three mice models were used as the foundation for this work. Embryonic tissue was analysed from wild-type CD1 and Lgr5-eGFP-IRES-CreERT2 mice, and adult intestinal tissue examined from tamoxifen-activated Villin-Cre-ERT2 Pten-/- Brafv600E mice. Culture of wild-type embryonic mouse intestine with and without segments removed and / or reversed was performed to investigate the question of what triggers the proximal-to-distal wave of villus morphogenesis. Immunohistochemical interrogation using anti-GFP antibodies was used in the Lgr5- GFP mice to identify the location of Lgr5-expressing cells during the development of villi. Bright-field microscopy, time-lapse in-incubator microscopy, and histological sections assessed villus morphology. The Villin-Cre-ERT2 Pten-/- Brafv600E mouse mutant was explored regarding the intestinal epithelial morphometric changes that occur following tamoxifen-induction. Abstract: Results The proximal-to-distal wave of villus morphogenesis was observed both in vivo and in vitro. Villus morphogenesis commences at embryonic day 14.5 in vivo and after three days in culture from e11.5 in vitro. The villus structures formed in vitro are significantly attenuated compared to in vivo development. An attempt was made to overcome this by providing intestinal explants with a blood supply to aid growth. Evidence is presented that suggest the proximal-to-distal wave of villus morphogenesis is driven by location specific factors intrinsic to each part of the bowel, rather than a domino-like signalling cascade travelling along the intestine. Lgr5-expressing intestinal stem cells were present in early development. Prior to villus morphogenesis they were uniformly distributed along the luminal surface of the intestinal epithelia. During the intense proliferation associated with villus morphogenesis they progressively congregated to the inter-villus spaces. Once villi are fully formed they were absent from the villi but identified in the inter-villus spaces. The Pten/Braf mouse mutant demonstrates villus morphological changes similar to those found following post-bowel resection adaptation. This suggests that there may be a role for Pten/Braf in the epithelial proliferation following extensive bowel resection. Signalling factors in these pathways may be candidate intestinotrophic agents for the treatment of short bowel syndrome. Abstract: Conclusions Before any processes that manipulate intestinal epithelia can be safely translated into therapies to aid adaptation in patients with intestinal failure, it is important to have a full and detailed understanding of the basic science principles that underpin the behaviour of the epithelial cells, both during development and in adulthood. I have explored and quantified the process of villus morphogenesis in the embryonic mouse, investigated the timing of appearance of Lgr5 intestinal stem cells, and interrogated a genetic mouse model with morphometric changes similar to those seen following small bowel resection. I propose two candidate intestinotrophic agents that may hold regenerative potential to augment post small bowel resection adaptation. The next stage of investigation would be to use a mouse model of small bowel resection with manipulation of cell signalling factors to assess impact on post resection adaptation. The ultimate goal would be to investigate epithelial activity in human neonatal intestine and explore methods of modulating this to improve the outcomes from post bowel resection intestinal failure.

In the central nervous system, the axons of neurons are protected from damage and aided in electrical conductivity by the myelin sheath, a complex of proteins and lipids formed by oligodendrocytes. Loss or damage to the myelin sheath may result in impairment of electrical axonal conduction and eventually to neuronal death. Such demyelination is responsible, at least in part, for the disabling neurodegeneration observed in pathologies such as Multiple Sclerosis (MS) and Spinal Cord Injury. In the regenerative process of remyelination, oligodendrocyte precursor cells (OPCs), the resident glial stem cell population of the adult CNS, migrate toward the injury site, proliferate and differentiate into adult oligodendrocytes which subsequently reform the myelin sheath. Existing research indicates that OPC migration is directed by chemomigratory signals released from the site of injury and that the absence of OPCs is a feature of some MS lesions, suggesting that increased recruitment of OPCs to injury sites might improve remyelination, eventually leading to treatments of patient pathologies. I hypothesized that as yet undiscovered migration cues for OPCs might be released at sites of demyelination, diffuse through the CNS tissue, activate distal OPCs and guide them back to sites of demyelination. In this thesis, I performed bioinformatics analysis of gene expression arrays and identified upregulated cell surface receptors on OPCs activated in a cuprizone model, and upregulated secreted factors in whole lesion sites from an LPC induced MS type injury model and a Spinal Cord Injury model. I then optimised the X-celligence system for the quantification of OPC migration in response to secreted factors identified in my bioinformatics screen. By combination of these techniques with immunofluorescent staining I discovered novel expression of the cell surface receptor CX3CR1 on OPCs, increased expression of the corresponding ligand CX3CL1 in both MS type injury and Spinal Cord Injury, increased directional migration of OPCs in response to low concentrations of CX3CL1, and increased maturation of OPCs into adult oligodendrocytes at high concentrations of CX3CL1. Taken together these results propose a system in which an increasing gradient of CX3CL1 released from the site of injury directs the recruitment, then maturation of OPCs, making CX3CL1 a master regulator of OPC led CNS regeneration.

It has been repeatedly proven that cell therapies can address many current unmet clinical treatment needs and also improve on current treatment options for various diseases, from neurological disorders to bone repair (Rosset et al. 2014; Corey et al. 2017). Though the potential of cell therapies has been demonstrated at a relatively small scale, the realisation of bringing cell based treatments to a larger market is hindered by the complexity of the product along with safety concerned and high production cost. Safety concerns can be informed with more in-depth analytical analysis of the product, however this in turn increase the costs involved in producing a cell therapy (Davie et al. 2012). Consequently the cost of analytical techniques also needs to be reduced, to address this need the area of microfluidic based bioanalytics holds much promise (Titmarsh et al. 2014). The culturing of human mesenchymal stem cells (hMSC) was used as a proof of concept model to demonstrate where improved bioanalytical and bioassay methods could be utilised in the production of cell therapies. Cells from four donors were cultured under three different oxygen environments and the conditioned medium assessed for pro-angiogenic capabilities using a tube formation bioassay and a proportion of the cytokine secretome profile measured using Luminex technology. Thorough secretome analysis it was shown that predicting cytokine levels based solely on the donor was not possible as the handling of the cells also had an influence on the secretome profile. The donor expression profiles did not behave in the same manner across all oxygen environments, for example in some donors IL-8 levels increased per cell at lower oxygen where as other donors showed a decrease per cell. While the tube formation assay showed some differences between donors in pro-angiogenic capabilities it also highlights the challenges with interpreting large data sets. The feasibility of using a microcapillary film (MCF) based enzyme-linked immunosorbent assay (ELISA) to detected two relevant cytokines, IL-8 and hepatocyte growth factor (HGF) was investigated. Following on from this work the development of a combined MCF ELISA assay with hMSC cell culture to produce a fully closed cell screening system was initiated. It was shown that it was feasible to measure IL-8 and HGF using the MCF ELISA platform but further work would need to be done to make the system more compatible with the manufacturing environment. In order to adapt the MCF to also be an hMSC culture platform the first challenge was to functionalise the Fluorinated Ethylene Propylene (FEP) surface of the MCF. It was concluded that a poly (vinyl- alcohol) (PVA) and gelatin mixture produced a homogenous coating to which a consistent level of hMSC would attach. This work was carried out on a flat surface; therefore steps were taken to adapt this knowledge into the MCF, while there was evidence of hMSCs present inside the MCF more work will need to be done to bring this concept to an established platform.