RICERCA ITALIANA     

Cell therapy: development of biomedical and pre-clinical models using non laboratory animals

Università degli Studi di Milano

Abstract

A stem cell can both self-renew, in an undifferentiated state, and differentiate into one or more cell types. Stem cells are defined as totipotent or pluripotent when can originate all or most cell types that compose an organism, as is the case in the early embryo. Stem cells exist also in the adult, these cells however can give rise to a limited number of cell types and are defined as multipotent.
Stem cells hold a great potential both for research and clinical applications. Whereas studies on basic mechanisms are better performed with mouse cells, clinical applications would greatly benefit by the development of alternative animal models to be used beside the mouse which could provide information more relevant for the human species.
The use of traditional laboratory animals (mouse, rat, rabbit) needs to be followed by pre-clinical trials based on experimental models whose morphology, physiology and genetic background are more similar to humans. Pig and sheep have been used for many years as suitable models in different branches of medicine. Therefore it is necessary to develop these models also in the field of stem cell therapy deriving stem cells of different origins in these species.

Aim of this project is the derivation and characterization of pig and sheep stem cell lines in order to use them in pre-clinical trial for the development of cell therapy for human and veterinary applications.

The four units involved in the project will work on pig and sheep embryonic stem cells, sheep primordial germ cells and sheep adult mesenchimal bone marrow cells.

Based on preliminary results obtained by the different units, the project will define the procedures required for stem cells derivation from different sources analyzing, testing and adapting parameters and procedures currently available for mouse and human cell lines.
This preliminary phase will be followed by the identification of cellular and molecular markers defining pluripotency in the considered species. This will enable the definition of the optimal condition for obtaining and maintaining stable cell lines that can be compared to those currently available of human and mouse origin. Subsequently, stem cell plasticity will be evaluated studying the response of the different cell lines to biochemical stimuli as well as to the interaction with other cell types both in vivo and in vitro.

The results of this research will provide important information enabling the characterization of cell differentiation in these species. Due to the significant differences already highlighted between human and mouse cell lines the data that will be obtained during this project will make it possible to evaluate whether and to what extent pig and sheep models will enable substantial progress towards the application of cell therapy in human and veterinary medicine. <<<

Principal Investigator

Fulvio Gandolfi Università degli Studi di MILANO

Research Objectives

Aim of the present project is to derive pig and sheep stem cell lines of different origin in order to progress toward the clinical application of cell therapy in human and veterinary medicine.

There different kind of stem cells will be derived and characterized:
• pig and sheep embryonic stem cells
• sheep primordial germ cells
• sheep adult mesenchimal stem cells

For each cell line, aim of the project is:
• to improve the derivation procedures
• to evaluate the degree of self renewal through the analysis of specific markers
• to optimize the culture protocols for the establishment and maintenance of stable cell lines
• to identify the procedures for obtaining controlled cell differentiation in vitro
• to determine the degree of cell differentiation and integration following in vivo transplantation <<<

Timescale

24 months

National and international background

A stem cell can both self-renew, in an undifferentiated state, and differentiate into one or more cell types [1]. Stem cells are defined as totipotent or pluripotent when can originate all or most cell types that compose an organism, as is the case in the early embryo. Stem cells exist also in the adult, these cells however can give rise to a limited number of cell types and are defined as multipotent [2].
Their possible application to cell therapy has recently generated great excitement [3]. Stem cells transplanted into a suffering organ have been shown to proliferate, give rise and replace the various damaged cell types and restore the original functions. Experiments carried out in laboratory animals have demonstrated stem cell ability to regenerate complex anatomical structures such as the brain [4] and the heart [5]. These observations have opened the way and given new expectations in the management of a number of lethal pathologies like Parkinson’s and Alzheimer’s diseases [6] or heart failure [7].

Stem cells can be of different origin

1. Embryonic Sten cells (ESC)

Embryonic stem cells are derived from embryos at the blastocyst stage, as originally shown in the mouse [8, 9] and recently demonstrated in human [10, 11]. Stem cells obtained in this way possess the highest possible differentiation plasticity and may be induced to differentiate into almost all cell types [12, 13]. Isolation of ES cells in species other than the mouse has generally proven problematic and, even in mouse, it actually works efficiently only with embryos of inbred 129 and C57BL/6 strains [14]. Thus there is a strong genetic component to ES cell derivation. An ES cell retains the essential features and identity of an epiblast cell. Nonetheless, it is altered in certain respects, such as dependence on cytokines and fidelity of imprinting, from an embryonic cell in situ. In reality, ES cells should be considered a cell culture phenomenon or even an artefact. From this perspective it is not surprising that there are significant differences between species, reflecting not the intrinsic status of the epiblast but its capacity to adapt to an arbitrary set of artificial conditions. Therefore, the phenotypes that may survive or emerge in culture will not necessarily be invariant between different genotypes.
This concept has become truly evident when primate [15] and human [10] ESC have been derived. This not only opened the way for a therapeutic use of ESC but it also clearly showed that several differences exist between human and mouse ES cells [14, 16].
In fact the specific factors currently identified that sustain mouse ES cells do not support human ES cells. Leukaemia inhibitory factor and related cytokines fail to support human or nonhuman primate ES cells in media that support mouse ES cells [10, 17]. Recent studies show that bone morphogenetic proteins (BMPs) combined with leukaemia inhibitory factor support mouse ES cells in serum-free conditions, yet BMP treated human ES cells rapidly differentiate in conditions that would otherwise support self renewal, and suppression of BMP signalling is beneficial to human ES cell culture if this activity is present [18].
Another important issue to be borne in mind with human cells is the likely impact of genetic heterogeneity as opposed to mouse ES cells which are predominantly derived from completely inbred embryos of strain 129. Finally, reproducing early human differentiation processes in vitro may prove more problematic than for mouse ES cells. The formative processes of gastrulation, primary tissue specification, and organogenesis take place over approximately 4 days in the mouse embryo and are well described. The corresponding window of human embryogenesis, takes place between 2 and 5 weeks after fertilization, and is inaccessible. [14].
Therefore it looks rather clear that it is not possible to directly extrapolate to the human species the findings obtained from mouse studies. As a consequence, whereas in vitro experiments can easily be performed with human ESC in order to determine the species-specific aspects, it would be highly desirable that a suitable animal model be available for pre-clinical studies involving in vivo ESC transplantation.


2. Cellule germinali primordiali (PGC)

Totipotent and pluripotent stem cell lines can also be derived from primordial stem cells [19]. In these years many studies have been carried out in mouse developing systems to maintain PGCs at undifferentiated stage in culture, stimulate their replication in vitro and verify their toti e pluripotency, by generating chimeric animals or developing tumors with allografting [20-22].
However, even if some interesting results have been obtained, different problems have been observed with a consequent limitation of their potential use in clinical applications. Despite these problems the derivation of stem cell from primordial germ cells (EG cells) represents an important alternative because they have potentially a different epigenetic status compared to stem cells derived from preimplantation embryos (ES cells). Recent studies showed that epigenetic status of stem cells could significantly affect the success of cell therapy. Moreover, PGCs in culture are important tools for studying factors involved in their survival, proliferation and differentiation into gametes precursors with important clinical relevance. For example, testicular cancer in men arises from carcinoma "in situ" cells, which probably originate from PGCs that escaped normal differentiation process.

3. Adult stem cells

Stem cells also exist in the adult individual, separated in special environments called niches. Beside well-known stem cells districts like bone marrow, intestinal crypts and the basal layer of the epidermis, stem cells have been identified also in organs where cell renewal was not previously recognized such as the central nervous system and the skeletal muscles. These cells are able, not only to replace units of the same tissue but also to generate different cell types, albeit with a more limited plasticity compared to their embryonic counterpart [2].
The unexpected discovery of the differentiation plasticity of stem cells collected from organs of adult individuals [23, 24], initiated a large number of experiments aimed at making of “adult stem cells” the politically correct therapeutic tool for regenerative medicine. Indeed, a plethora of scientific contribution regularly appear on the pages of the best science journals [25, 26]. Moreover, adult stem cells research is rapidly translated into clinical practice, as witnessed from many pilot studied currently on going in USA and Europe [27]. Two recent studies however suggest a note of caution on adult stem cell transplantation. The studies, carried out on mice by two independent teams, demonstrated that haematopoietic precursors transplanted into experimentally infarctuated hearth do not differentiate into cardiomiocytes, as expected, but rather acquired a fibroblast phenotype, which could actually induce a fibrotic transformation of the original lesion [28, 29]. These, and other published data [30, 31], brought about some scepticism on the real regeneration potential of adult stem

Irrespective of their different origin and of their specific advantages or disadvantages, it is crucial to acquire a thorough knowledge of the mechanisms regulating stem cell biology before these cells can be used for clinical application both in human and veterinary medicine.
Stem cells hold a great potential both for research and clinical applications. Whereas studies on basic mechanisms are better performed with mouse cells, clinical applications would greatly benefit by the development of alternative animal models to be used beside the mouse which could provide information more relevant for the human species.
Therefore the use of traditional laboratory animals (mouse, rat, rabbit) needs to be followed by pre-clinical trials based on experimental models whose morphology, physiology and genetic background are more similar to humans. Pig and sheep have been used for many years as suitable models in different branches of medicine. Therefore it is necessary to develop these models also in the field of stem cell therapy deriving stem cells of different origins in these species. <<<