6 research outputs found
New culture systems for mesenchymal stem cells
Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage
tissues of the human body. In addition, these cells can form muscles, ligaments and
neurons. This wide multipotency has made mesenchymal stem cells of particular
interest in the fields of tissue engineering and regenerative medicine. Furthermore,
mesenchymal stem cells can modulate the immune system by reducing factors that
increase inflammation and immune recognition. This immune recognition
suppression has resulted in their application as part of bone marrow transplantation
in the prevention of 'graft versus hostâ disease. There are hundreds of on-going
clinical trials using these cells for the treatment of autoimmune diseases such as type
I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells
has brought in to focus the culture methods used to for their expansion and
manipulation. Currently, animal derived components are used as surfaces for their
growth and as components in the culture media. This exposes these cells to animal
pathogens and antigens that can be passed to the recipients of these cells.
In the first part of this thesis, polymer microarrays were employed to identify
alternatives to the biological surfaces currently used for mesenchymal stem cell
culture. This platform allowed hundreds of polyacrylates/acrylamides and
polyurethanes to be simultaneously scrutinised to identify surfaces that could support
their growth and maintain their stem cell characteristics. Identified polymer surfaces
were monitored in long-term culture (10 passages) and were shown to retain the cell
phenotype and capacity to differentiate, thus providing chemically defined substrates
for long-term mesenchymal stem cell culture.
In the second part of this thesis, a 'smartâ polymer microarray of hydrophilic cross-linked
polymers (hydrogels) were used to remove another key biological component
of culture, trypsin. These 'smartâ hydrogels modulated their properties depending on
the temperature. Hydrogels that could trigger mesenchymal stem cell release after a
reduction in temperature were identified. A unique passaging system using a modest
temperature reduction for 1h was developed as a passaging method. Cells were
maintained and monitored for 10 passages using this novel enzyme free passaging
method. Analysis of the mesenchymal stem cell phenotype and differentiation
capacity revealed this method superior than conventional culturing methods.
In the final part of this thesis, a 'knowledge-basedâ small molecule library was
designed, which could potentially yield small molecules to manipulate/enhance the
mesenchymal stem cell state without the use of biological components. The key
protein pathways that control the stem cell state were examine with the
bioinformatics tool GeneGo was used to identify compounds that affected these
pathways, resulting in selection of 200 small molecules. The effect of the small
molecules on the mesenchymal phenotype was examined and 5 small molecules were
identified that enhanced the phenotype of these cells. The anti-inflammatory
properties associated with the hit compounds led to the investigation of their effects
on key surface proteins associated with the immune-modulatory state of the cells. In
this preliminary study, two of the small molecules, estriol and spermine, increased
the expression of a key mesenchymal stem cell marker STRO-1 and down regulated
ICAM-1, a critical component of the immune modulation capacity of this cell type
Long term Mesenchymal stem cell culture on a defined synthetic substrate with enzyme free passaging
Mesenchymal stems cells (MSCs) are currently the focus of numerous therapeutic approaches in tissue engineering/repair because of their wide multi-lineage potential and their ability to modulate the immune system response following transplantation. Culturing these cells, while maintaining their multipotency in vitro, currently relies on biological substrates such as gelatin, collagen and fibronectin. In addition, harvesting cells from these substrates requires enzymatic or chemical treatment, a process that will remove a multitude of cellular surface proteins, clearly an undesirable process if cells are to be used therapeutically. Herein, we applied a high-throughput âhydrogel microarrayâ screening approach to identify thermo-modulatable substrates which can support hES-MP and ADMSC growth, permit gentle reagent free passaging, whilst maintaining multi-lineage potential. In summary, the hydrogel substrate identified, poly(AEtMA-Cl-co-DEAA) cross-linked with MBA, permitted MSCs to be maintained over 10 passages (each time via thermo-modulation), with the cells retaining expression of MSC associated markers and lineage potency. This chemically defined system allowed the passaging and maintenance of cellular phenotype of this clinically important cell type, in the absence of harsh passaging and the need for biological substrates
Dielectrophoresis based discrimination of human embryonic stem cells from differentiating derivatives
Assessment of the dielectrophoresis(DEP) cross-over frequency (f xo), cell diameter, and derivative membranecapacitance (C m) values for a group of undifferentiated human embryonic stem cell (hESC) lines (H1, H9, RCM1, RH1), and for a transgenic subclone of H1 (T8) revealed that hESC lines could not be discriminated on their mean f xo and C m values, the latter of which ranged from 14 to 20âmF/m2. Differentiation of H1 and H9 to a mesenchymal stem cell-like phenotype resulted in similar significant increases in mean C m values to 41â49âmF/m2 in both lines (pâ<â0.0001). BMP4-induced differentiation of RCM1 to a trophoblast cell-like phenotype also resulted in a distinct and significant increase in mean C m value to 28âmF/m2 (pâ<â0.0001). The progressive transition to a higher membranecapacitance was also evident after each passage of cell culture as H9 cells transitioned to a mesenchymal stem cell-like state induced by growth on a substrate of hyaluronan. These findings confirm the existence of distinctive parameters between undifferentiated and differentiating cells on which future application of dielectrophoresis in the context of hESC manufacturing can be based
Expression of FBN1 during adipogenesis:relevance to the lipodystrophy phenotype in Marfan syndrome and related conditions
Fibrillin-1 is a large glycoprotein encoded by the FBN1 gene in humans. It provides strength and elasticity to connective tissues and is involved in regulating the bioavailability of the growth factor TGF beta. Mutations in FBN1 may be associated with depleted or abnormal adipose tissue, seen in some patients with Marfan syndrome and lipodystrophies. As this lack of adipose tissue does not result in high morbidity or mortality, it is generally under-appreciated, but is a cause of psychosocial problems particularly to young patients. We examined the role of fibrillin-1 in adipogenesis. In inbred mouse strains we found significant variation in the level of expression in the Fbn1 gene that correlated with variation in several measures of body fat, suggesting that mouse fibrillin-1 is associated with the level of fat tissue. Furthermore, we found that FBN1 mRNA was up-regulated in the adipose tissue of obese women compared to non-obese, and associated with an increase in adipocyte size. We used human mesenchymal stem cells differentiated in culture to adipocytes to show that fibrillin-1 declines after the initiation of differentiation. Gene expression results from a similar experiment (available through the FANTOM5 project) revealed that the decline in fibrillin-1 protein was paralleled by a decline in FBN1 mRNA. Examination of the FBN1 gene showed that the region commonly affected in FBN1-associated lipodystrophy is highly conserved both across the three human fibrillin genes and across genes encoding fibrillin-1 in vertebrates. These results suggest that fibrillin-1 is involved as the undifferentiated mesenchymal stem cells transition to adipogenesis but then declines as the developing adipocytes take on their final phenotype. Since the C-terminal peptide of fibrillin-1 is a glucogenic hormone, individuals with low fibrillin-1 (for example with FBN1 mutations associated with lipodystrophy) may fail to differentiate adipocytes and/or to accumulate adipocyte lipids, although this still needs to be shown experimentally. (C) 2016 The Authors. Published by Elsevier Inc