RICERCA ITALIANA     

Embryonic stem-like cells obtained from fibroblasts cultured in presence of mouse embryonic stem cell extracts

Università degli Studi di Pavia

Abstract

A wide variety of diseases that affect humans are associated with cell death. Twenty-first century medicine aims to make use of cell therapies to replace dead cells with new ones, as much as it is now taking advantage of organ transplantation. Stem cells represent the ideal biological source for the regeneration of damaged tissues and organs. Recent studies have demonstrated that when differentiated somatic cells are cultured in presence of cell extracts isolated from other cell types, they are induced to acquire a new state of differentiation.

The main objective of this research project is to obtain a large number of pluripotent stem cells by culturing differentiated somatic cells in mouse embryonic stem (ES) cell extracts.

Following culture of fibroblasts in presence of ES cell extracts, it is expected, from our preliminary results, that these differentiated cells will acquire a pluripotent competence.
Functional reprogramming of fibroblasts will be defined analysing:

A) The capacity of reprogrammed fibroblasts to:
1. differentiate into embryoid bodies;
2. partecipate to the formation of a new individual and its germ line following their transfer into blastocysts;

B) the nuclear architecture: localisation of constitutive heterochromatin and centromeres;

C) the methylation pattern of the entire genome and of single specific genes;

D) the expression of genes and proteins that mark the occurence of cell reprogramming. <<<

Principal Investigator

Silvia GARAGNA Università degli Studi di PAVIA

Research Objectives

Our working hypothesis is that pluripotent stem cells could be obtained in vitro by culturing differentiated somatic cells (i.e., mouse fibroblasts) in cell extracts obtained from mouse embryonic stem (ES) cells. We expect that the pluripotent stem cells obtained through this procedure would be capable of extensive proliferation in an undifferentiated status and would provide an unlimited source, following culturing in specific differentiating media, of different somatic cell types. These studies will constitute a sound background of knowledge necessary for the identification of the mechanisms and molecules responsible for biological processes like the control of cell de-differentiation, the establishment of pluripotency and the acquisition of a new differentiated state.

Although human ES lines have been established since 1998 (Thomson et al., 1998), the amount of knowledge and research tools (e.g., markers, methods for inducing the differentiation of ES cells etc.) gathered from 24 years of research on mouse ES cells (Evans and Kauffman, 1981; Martin, 1981) suggest that our working hypothesis should best and more quickly be first tested on this animal. The knowledge gathered from these studies represents a first objective towards the long-term goal of reproducing the capacity of ES cell extracts of reprogramming differentiated somatic cells, without the need of ES cells.

The achievement of the aims of this study will have an impact on:
1) basic science, providing the necessary knowledge for the identification of the mechanisms responsible for the control of cell dedifferentiation and the establishment of pluripotency;
2) medicine, after having been tested on model animal of human disease, the results will provide the methodology to produce an unlimited source of replacement cell types for therapeutic applications.


BIBLIOGRAPHIC REFERENCES

- Evans M., Kaufman M. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156, 1981.

- Martin G, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634-7638, 1981.

- Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-1147, 1998. <<<

Timescale

24 months

National and international background

A wide variety of diseases that affect human beings are associated with cell death. Twenty-first century medicine aims to make use of cell therapies to replace dead cells with new ones, as much as it is now taking advantage of organ transplantation. Stem cells (SCs) represent the ideal biological material for the regeneration of damaged tissues and organs.

SCs compartments are present in adult tissues and organs, such as the epithelium, liver, bone marrow, muscle, gut and central nervous system. Adult SCs are quiescent or slow proliferating cells but have the ability to resume proliferation activity to replace dead and/or injured cells. In some cases, both the original SCs and the cells to which they give rise derive from the same embryonic germ layer (intra-germ layer conversion) (Jackson et al., 1999; Mahmud et al., 2002; Mc-Kinney-Freeman, 2002; Orlic, 2004). However, striking examples of trans-germ layer conversion, in which SCs and their progeny belong to developmentally unrelated cell lineages, have been reported (Kopen et al., 1999; Sanchez-Ramos et al., 2000; Woodbury et al., 2000; Petersen et al., 1999; Wang et al., 2002; Bjornson et al., 1999; Galli et al., 2000; Munoz-Elias et al., 2003, 2004).

SCs isolated from the adult organism have already been used for the treatment of leukemia, limbal stem cell deficiency (Rama et al., 2001) and post-infarctual necrosis (Orlic, 2004). Very encouraging results have been obtained when using embryonic stem (ES) cells. These stem cells can extensively proliferate in an undifferentiated state and, when properly stimulated, may provide with an unlimited source of many types of differentiated somatic (for a review see Rippon and Bishop, 2004) and germ (Hubner et al., 2003) cells. ES cells have been successfully employed in animal models for the treatment of diseases such as Parkinson (Kim et al., 2002), immunodeficiencies (Rideout et al., 2002) and injuries of the spinal cord (McDonald 2004), although their use in human cell therapy is widely discussed because of ethical and technical reasons.

Alternative methods for obtaining totipotent/pluripotent cells have been developed.

Nuclear transfer experiments (the Coordinator of Research Unit N. 2 was part of the international team that has carried out the study that has led to the cloning of the first mouse, see Wakayama et al., 1998) have clearly shown that enucleated mammalian oocytes have the capacity of reprogramming nuclei of terminally differentiated somatic cells (for a review see Brem and Kuhholzer, 2002). The newly reconstituted clonote is capable of initiating and terminating embryonic development and, with a low percentage, some of these fetuses can reach birth and adulthood. Although it is unknown which are the mechanisms and molecules involved in the reprogramming process, these experiments demonstrate that the epigenetic modifications that allow differentiated cells to perpetuate their molecular memory (which garantees the cell to retain its identity) may be erased and the genome of a terminally differentiated somatic cell may acquire a totipotent or pluripotent state.
Nuclear transfer is a novel technique and has a low efficiency as success is obtained in only about 1-5% of the attempts (Brem and Huhholzer, 2002). Novel methodologies for an efficient functional reprogramming of high numbers of somatic cells into other cell types that would overcome difficulties associated with nuclear transfer, involve the transdifferentiation of somatic cells in culture. Cell fusion experiments have demonstrated that ES and embryonic germ (EG) cells show cellular reprogramming activity (Tada et al., 2001; Ying et al., 2002; Terada et al., 2002). Recent studies have demonstrated the possibility of obtaining transdifferentiation in culture: myoblasts transdifferentiate into mature adipocytes when cultured with adipogenic transcription factors (Hu et al., 1995); induction of a hepatic transcription factor in pancreatic cells also causes their conversion into hepatocytes (Shen et al., 2000); a block of gap-junctions between cultured osteoblasts leads to an adipocitic phenotype (Schiller et al., 2001); embryonic or neonatal umbilical vein endothelial cells transdifferentiate into cardiomyocytes when co-cultured with neonatal rat cardiomyocytes (Condorelli et al., 2001).

Turning point experiments have been published in recent papers (Hakelien et al., 2002, 2004; Landsverk et al., 2002; Gaustad et al., 2004). Cell extracts isolated from differentiated somatic cells of different nature were capable to reprogram gene expression in other somatic cell/isolated nuclei types. Fibroblasts exposed to extracts from human T cells or from a transformed T-cell line showed the activation of lymphoid cell-specific genes and expression of T-cell specific antigenes. When fibroblasts where cultured with neuronal precursor cell extracts, they expressed a neurofilament protein and extended neurite-like outgrowths. Human adipose tissue stem cells were reported to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract, indicating the possibility of transpecies efficient reprogramming of somatic cells (Gaustad et al., 2004).
This functional reprogramming of differentiated somatic cells is also evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodelling complex and changes in chromatin composition. The observed transdifferentiation involves many complex cellular changes, including epigenetic modifications of DNA methylation, chromatin composition and packaging which lead to the activation or silencing of specific genetic pathways. The functional reprogramming was reported to be stable through many cell divisions over several weeks of cell culture (Hakelien et al., 2002, 2004; Hakelien and Collas, 2002; Landsverk et al., 2002). However, the stability of cell reprogramming depended on the source of cell extracts: transient functional cell reprogramming was described after alteration of cell fate when fibroblasts where cultured in presence of insulinoma cell line extracts (Hakelien et al., 2004).

These data show the molecular dominance of a certain cell type over another, resulting in the reprogramming of the susceptible cell by the dominant one. The use of cell-free extracts for obtaining large quantities of transdifferentiated cells seems to be very promising and could be a powerful system for analysing cell reprogramming events as they occur in vitro. <<<