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Reference: Schneider MR, Adler H, Braun J, Kienzle B, Wolf E, Kolb H-J. Canine Embryo-Derived Stem Cells – Towards Clinical Relevant Animal Models for Evaluating Efficacy and Safety of Cell therapies. Stem Cells 2007;25:1850.

Summarized by: Ibrahim Odewale, Fall 2007

 

LAY SUMMARY

Embryonic stem cells (ESCs) can generate all types of cells, which could be used to replace damaged cells or to regenerate damaged tissues.  ESCs are of special interest because of their potential clinical application in treating diseases, and replacing pathological cells and tissues.  Currently, ESCs have been extensively studied in mouse; however, understanding the properties of these cells in other animal models is necessary, in particular the animal close to human is need for future development of technologies for humans. Canine (dog) ESCs also have some advantages over mouse ESCs. Canines have a longer life-span than mice; this will enable scientists to do long-term studies with ESCs that are currently impossible in mouse models.  Canine diseases are similar to human diseases; this similarity makes them a better model for human diseases.  The significant size difference between canine and mouse will enable scientist to study and anticipate potential problems when stem cell therapies are scaled up for human delivery.

This study confirms the possibility of creating ESCs from canine embryo, and the ability to induce differentiation into hematopoietic stem cell progenitors, blood forming cells.  The inner cell mass (ICM) was mechanically removed from canine blastocysts at the 8 cell stage.  The cells were cultured on mitotically-inactivated mouse embryonic fibroblast.  These cultured cells were found to exhibit the shape and arrangement characteristic of ESCs, and expressed ESC genes, such as NANOG, OCT4, SOX2 and SSEA-1.  The expression pattern parallels what is observed in mouse ESCs. When the cells were grown in suspension media, they formed embryoid bodies, which is a step following ESC development.  The embryoid bodies gave rise to various cell types when they are transferred to adhesive plates. 

Differentiation into blood forming cells was achieved by growing the ES-like cells on top of an irradiated stroma cells from OP9 mice. Differentiation was confirmed by probing for CD34, which is a marker for hematopoietic progenitor cells, and GATA2, a hematopoietic transcription factor.  Upon confirmation of the hematopoietic lineage the cells were further cultured to see if they will give rise to hematopoietic colonies.  Colony-forming units were seen nine days after the culture, addition of hematopoietic growth factors caused colony-forming units to form in 6 days rather than 9 days. 

 

SCIENTIFIC SUMMARY

Embryonic stem (ES) cells are can differentiate into cells of the three germ layers.  ES cells are of special interest because of their potential clinical application in treating diseases, and replacing pathological cells and tissues.  ES cells have the potential for tissue repair or regeneration. 

The study of ES cells in canine is important because mouse models do not adequately mimic all human diseases. Canines are long lived; therefore, long-term studies which are impossible in mouse models can be done in canine.  Larger animals such as canines also acquire diseases that are similar to human diseases; this similarity makes them a better model for human diseases.  The significant size difference between canine and murine will enable scientists to study and anticipate potential problems when stem cell therapies are scaled up for human. All the aforementioned reasons make ES cell study in canine an important part of stem cell research because the techniques used will prove to be more applicable to human subjects.   This summary outlines the attempt to establish canine ES cell lines, and the induction of the stem cells into hematopoietic progenitor cells.

Inner cell mass (ICM) was mechanically removed from canine blastocysts at the 8 cell stage.  The cells were then cultured on mitotically-inactivated mouse embryonic fibroblast.  They exhibited the characteristic ESC morphology, they also expressed pluripotency markers.   The cells were further subcultured in suspension culture, which resulted in the formation of embryoid bodies that further differentiated into various cell types, including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells (Figure 1).  The formation of these cell types confirms the pluripotency of the cells. The ES-like cells were also cultured independent of the mouse feeder layer, they where then characterized for both pluripotency markers.  Lack of differentiation was confirmed via alkaline phosphatase activity, and the following pluripotency markers; NANOG, OCT4 and SOX2.  The ES-like cells were also studied with antibodies against stage-specific embryonic antigen SSEA-1 and SSEA-4.  The cells were positive for SSEA-1 but negative for SSEA-4, which is in accordance with the expression pattern seen in mouse.  The cells were induced towards hemotopoietic progenitor cells by coculturing the cells with irradiated OP9 bone marrow stroma cells.  The cells were then subjected to fluorescence-activated cell sorting (FACS) analysis using anti-canine CD34 antibody.   Almost none of the cells stained positive on day 0 but more than half of the cells expressed CD34 at days 6 and 9.  RT-PCR was also done to show the gene expression level of CD34 and GATA2, a transcription factor for hematopoietic progenitor cells.   The expression of both genes mirrored the result of the FACS analysis with no expression on day 0 but substantial expression on days 6 and 9. 

The cocultured cells were harvested on day 0, day 6, and day 9, and then cultured in 6-well plates to see if they will form colony-forming units (CFUs) of the hemtopoietic lineage.  As expected the cells from day 0 did not grow into colonies, but cells from day 9 formed CFU-M, CFU-E, CFU-G, and CFU-GM.  Cells from day 6 formed colonies only when hematopoietic growth factors were added. 

Although, undifferentiated phenotype was only maintained through passage 8 and the possibility of having an unlimited culture of the differentiated cells is yet to be determined, this study further established the practicability of canine stem cell techniques.  These techniques can be used as a platform for further stem cell studies in canine models, or they can be carried over to other animal models.  This study will also play a role in potential clinical applications of organ and tissue regeneration in veterinary medicine.

 

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