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Autogeneic feeder cell system as support for the in vitro growth of human embryonic stem cells

(Stojkovic P et al, Stem Cells 2005;23:306-314)
Summary by Arpita Patel and Zhaoyu Sun

LAY SUMMARY
Human embryonic stem cells (hESC) are pluripotent cells isolated from the inner cell mass (ICM) of human blastocysts between days 5-8 of fertilization. Pluripotency is assigned to the hESCs based on their ability to develop into cells of all tissues. The pluripotency of hESCs provides these cells as potential sources for therapies. However, the method to expand the hESCs in the laboratory has problems for future use in patients. Of significance is the incorporation of foreign substances into the hESCs while they are cultured with feeder cells from another animal, namely mouse. In addition to the use of mouse feeder cells, scientists have also used other animal substances.  Therefore, the major problem does not lie in the ability of hESC to be expanded and maintained in the laboratory for prolonged time. The major issue could be summarized as the fear of introducing foreign infectious agents and/or other risk factors that are derived from the mouse feeder cells.

This paper reports on a better support system derived from the same hESCs that are intended for growth. The hESCs used in the studies were commercially available and are called `hES-NCL1 and H1’. In this system, the feeder cell layer was obtained by allowing spontaneous maturation of the hESCs into cells with feeder property, called fibroblasts-like cells. These newly developed feeder cells allow for effective long-term expansion of the hESCs. The expanded cells were genetically identical to the original hESCs indicating that the cells did not show evidence of genetic abnormalities after expansion.   

SCIENTIFIC SUMMARY
Human embryonic stem cells (hESC) are pluripotent cells derived from the inner cell mass (ICM) of the human blastocyst at day 5 to day 8 (Figures 1A and 1B: Figures were drawn by Aripta Patel). The method to maintain and expand hESCs by in vitro method while maintaining them in undifferentiated state requires feeder cells or other supplements such as fibroblasts. The common feeder cells are mouse embryonic fibroblast (MEF), SIM mouse embryo-derived thioquanine and ouabain resistant cells (STO), or human fetal, neonatal, and adult cells. The feeder cells are thought to detoxify the culture media, secret unique growth supplements, and also secrete other proteins to generate extracellular matrix.

To date, several feeder cell systems have been used with different efficiency in supporting the growth while maintaining the hESCs in undifferentiated states. There are disadvantages in the use of feeder cells for hESCs cultures: 1) Xenogenic and allogenic feeders have the risk of cross-transfer of pathogens and other unidentified risk factors that limit their medical applications; 2) The efficiencies among feeder cells and cell-free supplements are different; 3) The use of human feeder cells is unacceptable due to ethical concerns associated with their sources from aborted fetuses.

This hot topic reports on alternate use of feeder cells in lieu of the current techniques. The authors of this paper have reported a new feeder system that utilizes hESCs-derived fibroblast cells (hES-dF). Micro satellite analysis was used to confirm that the hES-dFs cells and their autogeneic hESC lines (hES-NCL1 and H1) have the same genetic origin.  
To test whether hESCs maintain their pluripotency when grown on autogeneic hES-dFs, the authors compared the growth of both fresh and cryopreserved hESC colonies on fresh and cryopreserved hES-dFs, as well as on Matrigel. Cell-free conditioned media from hES-dFs showed homogeneity in colonies with typical morphology of hESCs. The investigators determined whether allogeneic feeder cells also support undifferentiated growth of hESCs by switching the hES-dFs with those generated from another hESCs.

By RT-PCR the authors showed the expression of markers consistent for stem cells in the hESC lines that were placed in cultures with hES-dFs feeder cells. This was in contrast to the hES-dF feeder cells that showed high telomerase activity in the absence of stem cell markers. Karyotyping of both hESCs lines grown on autogeneic feeder cells were found to be normal. The pluripotency of the hESC lines grown on autogenic hES-dFs were demonstrated to retain pluripotency in SCID mice. The authors showed the development of teratomas of each germ layer after the hESC lines were injected into SCID mice. In addition, the authors showed the development of neuronal precursors, beating cardiomyocytes and endodermal cells in two of the studied ESC lines.

In summary, this report verified that autogeneic hES-dF cells could efficiently support and expand hESCs. The reported feeder cells that were derived from autologous hESCs are advantageous when compared with the model of using allogeneic feeder cells. The reported system eliminates the risk of transferring pathogens to the hESC, and provides a relatively safe method to expand hESC for therapeutic purposes.