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Bcl-2 could replace serum supplement and feeder-cell for the growth of mouse embryonic stem cell

(Yamane T et al, Proc Natl Acad Sci USA 2005;102:3312)
Summary by Marianne Castillo and Shakti H. Ramkissoon

LAY SUMMARY

Embryonic Stem Cells (ESCs) are cells that can be isolated from the inner cell mass of a blastocyst.  The blastocyst is a round mass of cells that make up the developing embryo at days 5-8.  When ESCs divide, the daughter cells (the two cells derived from the division) have the ability to take on different paths.  One of them can self-renew, meaning that it can form an identical copy of the ESC, while the other may differentiate, meaning that it can form the mature cells of the body.

ESCs are called pluripotent cells because they have the ability to differentiate into cells of the 3 germ layers:
             

1. Mesoderm - responsible for the formation of the cardiovascular system (heart and blood), the skeletal system (bones), etc.
2. Endoderm - responsible for the formation of the inner linings of organs, the lungs, the urinary bladder, etc.
3. Ectoderm - responsible for the formation of the skin and nervous system (brain and spinal cord).

The pluripotency of ESCs accounts for their importance as potentially therapeutic agents.  Once ESCs are isolated from a blastocyst, they can be grown and expanded in the laboratory in the presence of growth supplement known as Leukemia Inhibitory Factor (LIF). This factor is important for maintaining the pluripotency of ESC.  Additionally, serum and feeder cells play a supportive role in the nourishment of ESCs. In the absence of these feeder cells the ESCs lose their ability to remain as stem cells and can form different tissues.

The authors of this periodical report a new way to maintain and expand ESCs derived from mice in long-term cultures in the absence of serum and feeder cells.  This was accomplished by inserting the human gene, Bcl-2, into the mouse ESCs.  The major function of Bcl-2 gene is to protect cells from dying, including the ESCs. The paper has described the ability of ESC to survive with high levels of Bcl-2 without serum and feeder cells. On the other hand, the ESCs that did not have high levels of Bcl-2 could not survive in the serum- and feeder- free conditions. The researchers also noted that the ESCs with high levels of Bcl-2 were able to remain as stem cells when they were given the factor, LIF. This presence of this factor was critical since, in its absence, the cells proceeded towards maturation to form cells of other tissues rather than remaining as stem cells. The authors took steps to verify that their ESCs are really stem cells. There are many methods by which this question could be answered.  This paper opted to test if the ESCs could make cells that can form blood cells and brain cells. 

In summary, these results prove a method by which ESC could be expanded without the need for serum and feeder cells. 

Comments: Previously, human ESCs were maintained and expanded in culture using human serum and feeder cells; however, this proved to be an expensive method.  Other alternatives were implemented, such as the use of mouse serum and feeder cells. This alternative, however, led to the contamination and fusion of the human ESCs with the mouse feeder cells. Although, this paper provides a novel way to expand and maintain mouse ESCs in the laboratory in the absence of serum and feeder cells, we must consider the effects of inserting the human Bcl-2 gene into the ESCs.  Since Bcl-2 is a gene that prevents cell death, one needs to think about the potential cancerous effects it might have if these cells are used for cell therapy in the future.  

SCIENTIFIC SUMMARY
Embryonic stem cells (ESC) are pluripotent cells that are derived from the inner cell mass of blastocysts. The ESCs, like other stem cells undergo indefinite self-renewal in vitro. In the case of ESCs from mouse, these stem cells require leukemia inhibitory factor (LIF) and fetal bovine sera for growth. In the absence of these factors, layers of mouse feeder cells could maintain the ESC cultures. In serum- and feeder-free conditions, but in the presence of LIF, undifferentiated ESC numbers decline and differentiated cells increase.  These results suggest that factors provided by sera and/or feeders are required to fully support self-renewal, especially at sub-optimal culture conditions (low density). The premise is that these factors autocrine and paracrine survival/anti-apoptotic signals are minimal.  To further investigate the role of survival signals in the self-renewal of ESC, Yamana et al generated ES cell clones (ESC-BCL-2) expressing the anti-apoptotic protein Bcl-2 tagged with GFP. ESC-Bcl-2 cells cultured in the presence of serum and feeder conditions showed similar growth kinetics as control.  However, when cultured in the absence of serum and feeder conditions, parental ES cells showed increased Annexin V expression compared to ES-Bcl-2 cells, 46.7% and 14.7%, respectively.  Supplementation of ES-Bcl-2 cultures with LIF in the absence of serum and feeder conditions demonstrated that these conditions could adequately expand the ES-Bcl-2 cells while maintaining them as stem cells. To determine the multilineage potential of these expanded ES-Bcl-2 cells, in vitro differentiation studies were performed to generate hematopoietic colonies capable of producing mature myeloid cells.  Similarly, ES-Bcl-2 cells were able to generate neurons in vitro, as evident by the presence of neuron-specific markers.  For definitive confirmation of pluripotency, ES-Bcl-2 cells were injected into blastocysts and transferred to the uteri of pseudo-pregnant mice.  GFP-positive ES-Bcl-2 derived cells were detected globally in d11.5 embryos.  Tissue chimerism was found to be ~30% in all tissues analyzed by Sry gene expression in female offspring (ES-Bcl-2 cells were male derived).  Chimeric mice remained tumor free at 6 months of age.  Together these results describe a novel method to expand ES cells in the absence of serum and feeder conditions and yet maintain an undifferentiated/pluripotent state capable of forming all tissues of the body.