HESC METABONOMICS · Elucidation of Stem Cell Fate and Cell-type Characterization by 1H-NMR-based Metabonomics

Elucidation of stem cell fate and cell-type characterization by 1H-NMR-based metabonomics

Human embryonic stem cells (hESCs) show great potential for providing an ongoing source of cells for studying early human development, disease states and for applications in drug screening and regenerative medicine. However, directing stem cell differentiation in vitro has proved difficult with the result generally being the production of heterogeneous populations of cells at differing stages of differentiation. Mixed populations of cells are not useful for therapeutic application (undifferentiated hESCs can develop teratomas and teracarcinomas in vivo) and act as poor models for studying in vitro differentiation. Further study of hESC culture conditions and the pathways attributed to controlling stem cell fate are required. Metabolites act as substrates, enzymes and endpoints of biochemical pathways that functionally reflect cellular phenotype. Little is known regarding the metabolic changes that occur during hESC differentiation. Similarly, the role of metabolites in maintaining hESCs in vitro is poorly understood. This project addressed these deficits in knowledge by using a Nuclear magnetic resonance (NMR)-based metabonomics approach for examining metabolic changes associated with hESC stem culture and directed differentiation. As the project represented the Fellow's (and the Host's) first venture into metabonomics, careful training was undertaken using two sets of serum samples provided by collaborators within the host institution. This enabled the optimisation of 1D and 2D NMR experiments used later during the project but also provided an opportunity to gain skills in processing and statistical analysis of NMR data and analysis of metabolic pathways. These projects were very successful within their own right resulting in peer reviewed publications in high impact journals. NMR-acquired metabolic serum samples derived from patients suffering from Chronic lymphocytic leukaemia (CLL) could be used for rapid, accurate and early diagnosis of patients with an acute form of the disease. Early diagnosis means that more effective treatment regimes can now be designed. Positive results were also obtained in a collaborative project where blood metabolic profiling by NMR facilitated the discrimination of patients suffering cirrhosis from those with sub clinical minimal hepatic encephalopathy. Patients may now benefit from early treatment, which has been shown to improve outcome and quality of life. Culture media conditioned by human foreskin fibroblasts provide a complex supplement of protein and metabolic factors that support proliferation of hESCs in vitro. While some work has examined the protein complement of this media, detailed information regarding the metabolic component is lacking. The Fellow's work in this area has provided a detailed characterisation of metabolic factors present in conditioning media. The work shows that an NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is useful for monitoring hESC culture media preparation and optimisation. This should enable researches to develop more effective culture conditions required to realise the potential of hESCs in a therapeutic context. While the project initially aimed to examine metabolic changes associated with hESC differentiation induced by hypoxia, analysis of available results suggested the differentiation protocol was inconsistent. An alternative, more robust model was achieved by characterising metabolic changes during the differentiation of hESCs to motor neurons following an established protocol developed by collaborators within the host institution. This work identified various metabolic pathways activated and deactivated during hESC differentiation to committed motor neurons and provided exciting information that may be exploited to drive differentiation of hESCs to form more pure preparations of neuronal stem cells and motor neurons useful for therapeutic purposes.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз