Centre for Reproduction & Development

Reprogramming of somatic cells

Lab head: Dr Paul Verma

Pluripotent embryonic stem cells (ESCs) could potentially generate specific cell types for treating serious diseases. A major problem limiting the clinical use of ESCs is the potential for tissues derived from these cells to be rejected by receiving patients. The most attractive solution to this problem comprises transplanting tissues derived from ESCs genetically matched to each patient. Somatic cell nuclear transfer (SCNT), where an adult somatic cell is returned to a pluripotent state (a process called reprogramming) following transplantation to an enucleated oocyte, can be used to provide such cells, however, ethical and practical limitations associated with both oocyte donation and human SCNT raise serious concerns about the suitability of this method.

Alternative approaches to reprogramming cells include 1) fusion of somatic cells with ESCs and 2) introduction of a few key pluripotent genes into the somatic cells, also know as induced pluripotent stem cells (iPSCs).

Cell fusion
Little is known about the molecular mechanisms of fusion-based reprogramming or about the extent of somatic genes resetting in this system. Our group is investigating several critical issues related to fusion-based somatic cell reprogramming, such as control of fusion efficiency, changes in global and gene-specific DNA methylation, the role of cell cycle synchronization and the chromosomal stability in hybrid cells. Various somatic cell types are also being examined to determine whether various degrees of ”reprogrammability” exist which would identify appropriate target cell types for fusion-based reprogramming.

Diploidisation of reprogrammed cells
Somatic cells can be reprogrammed to an ES-like state following cell fusion to ESCs. This approach results in a tetraploid cell which contains the DNA from both the ESC and the somatic cell and therefore is not appropriate for clinical application. Our group is developing unique methods of returning fused somatic cells to a diploid state to overcome this. One such approach consists of first generating tetraploid ESCs by cell fusion of two diploid ESCs. The tetraploid ESCs are then fused to diploid somatic cells to form heterokaryons (two separate nuclei within a cell sharing common cytoplasm). Once the somatic nucleus is reprogrammed, the heavier tetraploid ESC nucleus can be removed by centrifugation. A proof-of-concept for this technique is outlined in Pralong et al 2005.

Isolation and characterization of bovine ESCs
The isolation and in-vitro maintenance of bovine embryonic stem cell lines is important for overcoming some of the difficulties related to production of transgenic cattle, and may significantly improve cloning efficiency in the bovine.

The establishment of pluripotent embryo-derived cell lines from the bovine, however, is more complex and less efficient than in mice. As part of a CRC funded project, we have examined several critical points influencing success in isolation of bovine ESC lines. We have identified that while heterogenous ES-like cell populations can be produced relatively easily, the establishment of large populations of homogenous bovine ESCs require new approaches. We have identified novel methods, protected by filed patents, to isolate and maintain bESCs with greater efficiency and to increase the number of ESCs obtained from a single embryo. Our current research is aimed at characterizing the bESCs and investigating their in-vitro and in-vivo differentiation potential. We are also exploring the utility of these new methods to isolate ESCs in other mammals.