Pluripotency, reprogramming and differentiation

Bertone group figureMalignant brain tumors are driven by abnormal neural stem cells. (A) GNS cells propagate indefinitely in culture and can differentiate into the major cell types of the central nervous system, such as astrocytes and oligodendrocytes. (B) Array CGH and genome resequencing identify chromosomal abnormalities and the disruption of genes affected by them. Detailed analysis of these cells from independent glioblastoma cases identified a molecular signature strongly correlated with patient survival (C), such that an increase in primary tumor biopsies is associated with more limited prognoses (D).

We investigate the cellular and molecular attributes of embryonic and tissue-specific stem cells using a combination of experimental and computational methods. We develop and apply genomic technologies to the analysis of stem cell function to address fundamental aspects of development and disease. Embryonic stem (ES) cells are similar to the transient population of self-renewing cells within the inner cell mass of the pre-implantation blastocyst (epiblast), which are capable of pluripotential differentiation to all specialised cell types comprising the adult organism. These cells undergo continuous self-renewal to produce identical daughter cells, or can develop into specialised progenitors and terminally differentiated cells. Each regenerative or differentiative cell division involves a decision whereby an individual stem cell remains in self-renewal or commits to a particular lineage. The properties of proliferation, differentiation and lineage specialisation are fundamental to cellular diversification and growth patterning during organismal development, as well as the initiation of cellular repair processes throughout life.

The fundamental processes that regulate cell differentiation are not well understood and are likely to be misregulated in cancer. A second focus in the lab is the study of neural cancer stem cells derived from human glioblastoma multiforme tumors. Using neural stem (NS) cell derivation protocols, it is possible to expand tumor-initiating, glioblastoma-derived neural stem (GNS) cells continuously in vitro. Although the normal and disease-related counterparts are highly similar in morphology and lineage marker expression, GNS cells harbor genetic mutations typical of gliomas and give rise to authentic tumors following orthotopic xenotransplantation. We apply genomic technologies to determine transcriptional changes and chromosomal architecture of patient-derived GNS cell lines and their individual genetic variants. These data provide a unified framework for the genomic analysis of stem cell populations that drive cancer progression, and contribute to the molecular understanding of tumorigenesis. 

Future plans

We have in place the most robust and stable systems for stem cell derivation and propagation, where controlled experiments can be performed in well-defined conditions. These assets are particularly valuable for studying cell populations that would normally be inacessible in the developing embryo. To realize the potential of ES cells in species other than mouse, however, precise knowledge is needed of the biological state of these cells, and particularly of the molecular processes that maintain pluripotency and direct differentiation. We are working to translate the knowledge and methods that have been successful in mouse ES cell biology to other mammalian species. This involves the characterization of germline-competent ES cells from the laboratory rat, along with the production of pluripotent human iPS cells using alternative reprogramming strategies. Through deep transcriptome sequencing we have shown a broad equivalence in self-renewal capacity and cellular state, albeit with intriguing species-specific differences. Thus, while ground-state pluripotency can be captured and maintained in several species, the mechanisms used to repress lineage differentiation may be fundamentally different.

Tumor-based cancer studies are limited by a number of factors, including cellular diversity of tissue biopsies, lack of corresponding reference samples, and inherent restriction to static profiling. Cancer stem cells constitute a renewable resource of homogeneous cells that can be studied in a wide range of experimental contexts and provide key insights toward new therapeutic opportunities. To this end we are carrying out in-depth analyses of our GNS cell bank using comprehensive genetic and transcriptomic profiling. With these data we are developing methods to stratify glioblastoma classes based on the molecular attributes and differentiation capacities of tumor-initiating stem cells. Existing tumor subtypes are associated with diverse clinical outcomes and therefore important for prognostic value, but as with any analysis of complex tissues, previous results have suffered from sample heterogeneity. With access to the underlying stem cell populations derived from parental tumors, we are refining existing subtype classification to improve the diagnostic utility of this approach. We are also performing functional experiments to identify alterations in GNS cells that impart tumorigenic potential. 

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