Mouse embryonic stem cells : The establishment of the system to produce differentiated cell types in vitro

1 Postgraduate Program of Veterinary Sciences (Programa de Pós-Graduação em Ciências Veterinárias, Faculdade de Veterinária FAV ET), Universidade Federal do Rio Grande do Sul (UFRGS). 2 Department of Physiology, Technical University Munich-Weihenstephan, Germany. 3 Group of Molecular and Functional Microbiology, Department of Molecular Biology and Biotechnology, Biotechnology Center UFRGS , RS, Brazil. 4 Department of Molecular Animal Breeding and Biotechnology, Ludwig Maximilian University Munich, Germany. CORRESPONDENCE: E. O. Cirne-Lima [e-mail: bcirne@dna.cbiot.ufrgs.br; Fax +55 51 3316 7305] Setor de Imunologia/DPCV/FAVET UFRGS. Caixa Postal 15094; 91501-970 Porto Alegre, RS Brazil. Received: January 2003 Accepted: March 2003 ABSTRACT

There are three types of stem cells: totipotent cells derived from the embryo, which can generate an entire organism and differentiate into all types of tissue.
Secondly pluripotent cells which can differentiate into cells of all three germ layers, but not develop to another complete individual.In the beginning pluripotent cells were isolated from teratocarcinomas, a complex type of malignant tumor containing a mixture of differentiated cell types, including derivatives of the three germ layers, and a number of undifferentiated cells, which are called embryonic carcinoma (EC) cells due to their similarity of early embryonic cells [13].EC cells are a very good model of multilineage differentiation, but their use as a developmental model is undetermined because of their aneuploid karyotype.
Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self renewal and a broad differentiation plasticity [16].Since the first ES cell lines (sharing many features with the EC) were established from the inner cell mass (ICM) of mouse blastocysts [8] others have been derived from eighth cell embryos or dissociated from morula.ES cells are a good model for development studies, especially because ES cells do not only differentiate in vitro and in vivo, but also can be propagated as a homogeneous uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype.
Considering that this cells can be propagated with the same characteristics it can be transfected with specific gene sequences and the transgenic lines obtained should be used as a model to study the influence of the expression of that specific genes on in vitro and in vivo tissue differentiation and furthermore this transgenic ES cell lines could be injected in an embryo to analyze the influence of the expression of that specific sequence upon embryo and/or animal development.
In addition, the nucleus of an embryonic stem cell transfected with a target gene or not could be injected by micromanipulation into an enucleated zygote to produce an animal clone.
The third group are the multipotent somatic stem cells, which are derived from adult, differentiated tissues and do not present the same plasticity, capacity of long-term propagation and less ability to generate different cell types.For example haematopoietic stem cells (HSCs) isolated from the bone marrow represent only a very small amount of around 1 in 15 000 cells [24].Beside the bone marrow cells, other adult tissues such as blood, brain, spinal cord, skeletal muscle, and epithelia of the skin and digestive system contain a similar type of multipotent somatic or adult stem cell population like the HSCs culture.But the main difference between embryo-derived pluripotent cells and soma-originated multipotent cells is that the latter are no longer able of generating germ cells and may also not differentiate into the wide range of cell types that can be derived from embryonic stem cells [5].
At the present report we decided to work with murine ES cell lines to induce in vitro differentiation into cardiogenic, myogenic and neurogenic cells using specific protocols established during the last years.

Cell lines
The mouse embryonic stem cell lines D3 [4] and R1 [14] were cultured in an undifferentiated state on feeder layer cells of primary mitomycin-treated embryonic mouse fibroblasts in DMEM (Dubco's modified Eagles medium, GibcoBRL Life Technologies, GmbH) supplemented with 15% FCS, 1% non essential amino acids, 0.1% β-mercapto ethanol, 1% L-glutamine and 1% LIF as described by Wobus et al. 1984 In vitro differentiation.To initiate in vitro differentiated ES cells were passaged onto gelatin-coated plastic petri dishes 24h before further treatment and cultured without LIF for 12h.Then ES cell colonies were disaggregated using trypsin solution (0.2% Gibco BRL in PBS, phosphate-buffered saline) and counted using a Neubauer's chamber.Depending on the supposed direction of differentiation between 400 and 600 ES cells/20 µl were cultured in hanging drops for two days.Within this period the cells aggregate and generate socalled "embryoid bodies" (EB).During this period we used DMEM culture medium supplemented with 15% of DCC-FCS (FCS treated with dextrancoated charcoal to remove endogenous retinoids and hormones).Afterwards EBs were cultured in suspension using bacteriological petri dishes for additional 3 days.Subsequently EBs were plated individually in 24-well plates, in DMEM supplemented with 15% fetal calf serum (FCS; selected batches, Gibco), 1% L-glutamine (Gibco, 2 nM), 0.1% β-mercaptoethanol (Serva, Heildelberg; final concentration 5x10 -5 M) and 1% nonessential amino acids (NAA; Gibco) as described [26,11].
Neurogenic differentiation: D3 ES cells, Iscove's medium supplemented as described above to the D-MEM plus retinoic acid (RA) 10 -7 M, daily supplemented during the first 3 days of 7 days suspension culture.Cell density: 800 cells/drop.Myogenic differentiation: D3 ES cells, D-MEM supplemented as described above.Suspension culture for 5 days in total, while cell density in hanging drops was 800 cells/20 µl.

RESULTS
The ES cell lines cultured under specific, well defined differentiation-inducing conditions developed from an undifferentiated stage resembling pluripotent cells of the early embryo into terminally differentiated stages of cardiomyocytes, myocytes, and neuronal cells.These cells were analyzed by cell morphology and also physiological properties, e. g. the in vitro differentiated cardiomyocytes resembled characteristics of atrial, ventricle, spontaneously beating.
It was found that parameters like number of cells in hanging drops, media and supplementations, ES cell lines and the time of EB plating influenced the developmental potency and differentiation features of ES cells in culture.
In this study we have successfully established for the first time in Brazil the embryonic stem cell technology including prolonged culture of various pluripotent ES cell lines as well as the directed induction of in vitro differentiation into a variety of specialized cell types like myogenic, cardiac and neuronal cells.
The present results gave us conditions to develop the ES cells routine in our University in order to apply this new technology to other projects where we will have conditions to analyze the influence of specific genes expression in tissue and animal development as well as we can collaborate with the groups of our University that dominate the animal reproduction technology to produce transgenic animals that should function as bioreactors to produce specific proteins.
Although the recent success in establishing human ES cells raised numerous public discussions concerning ethical, legal and political issues of this technique based on the idea to manipulate human embryos in order to introduce therapeutical cloning of human embryos used as sources of undifferentiated cells which subsequently will be used to generate autologous specific cell types in vitro for cell transplantation without any risk of rejection or any other side effect.Currently at the moment this topic is discussed very intensively and controversially around the world and it is necessary to define limits based on public and scientific local agreements.