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Keywords:

  • Embryonic stem cells;
  • In vitro differentiation;
  • Dog;
  • Hematology

Pluripotent embryonic stem (ES) cells, which are available in mouse [1, 2] and human [3], are permanent cell lines that can differentiate into cell types of all three germ layers. Therefore, ES cells have a remarkable potential for both basic research and clinical applications toward replacement of degenerated or malignant cells. From the perspective of hematology, where stem cell therapies are most advanced, ES cells have a number of advantages over conventional sources of transplantable material. They can be expanded indefinitely in vitro, and, more importantly, they can be obtained from a bank representing major haplotype combinations [4] or may even be derived by reprogramming somatic cells from individual patients. Proof of principle for this “therapeutic cloning” concept has been provided in the mouse [5]; however, translation of ES cell-based therapeutic strategies to clinical application requires larger animal models for predictive efficacy and safety studies.

The dog has been used for preclinical studies of stem cell transplantation, and many techniques have been derived from canine studies [6]. Furthermore, techniques applicable to canine cells and dogs can be applied to man, taking into account known biologic properties of the dog [7]. Since the dog is the ideal preclinical model for testing new therapies for many human diseases, the availability of canine embryo-derived stem cells for in vitro differentiation studies will be of great value for the development of new therapies, especially in hematology. Moreover, nuclear transfer from canine somatic cells is possible [8], providing the opportunity to evaluate the concept of “therapeutic cloning” in a clinically relevant animal model.

In a recent report, Hatoya and colleagues [9] describe the isolation of two ES-like cell lines from canine blastocysts. The cell lines were shown to exhibit characteristic ES-like morphology and expression of pluripotency markers. Importantly, the cells formed embryoid bodies in suspension culture, which differentiated upon adhesive culture into various cell types, including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells, demonstrating that these cells are indeed pluripotent. Unfortunately, it was not possible to maintain the undifferentiated phenotype of the cell lines beyond passage 8.

We have performed similar studies that confirm the possibility of establishing canine embryo-derived cell lines and, more importantly, demonstrate for the first time that these cells can be differentiated into hematopoietic stem cells. Eight blastocysts were obtained from a Golden Retriever bitch by flushing the uterine horns after ovariohysterectomy. After mechanical removal of the embryonic coats, the blastocysts were cultured individually on mitotically-inactivated mouse embryonic fibroblasts in 48-well plates (for further details, see supplemental online Methods). From one blastocyst, colonies exhibiting typical ES-like morphology (Fig. 1A) were obtained. At this stage, the cells could be maintained independent of mouse feeder cells but formed autologous feeders as previously described for human ES cells [10]. Cytogenetic analysis confirmed the canine origin of the cells, which contained a Y chromosome (Fig. 1B). The ES-like cells exhibited alkaline phosphatase activity (Fig. 1C) and expressed NANOG, OCT4, and SOX2, the most important pluripotency-associated transcription factors for mouse and human ES cells (Fig. 1D). Sequencing confirmed that the obtained polymerase chain reaction (PCR) products correspond to canine sequences. Furthermore, in agreement with the report by Hatoya et al. [9], the canine ES-like cells showed expression of SSEA-1 but were negative for SSEA-4 (data not shown).

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Figure Figure 1.. Canine embryo-derived stem cells. (A): Characteristic ES-like morphology. (B): Karyotype analysis indicating the presence of a Y chromosome. (C): Alkaline phosphatase activity. (D): Reverse-transcriptase polymerase chain reaction (PCR) showing mRNA expression of pluripotency markers NANOG, OCT4, and SOX2. (E, F): Fluorescence-activated cell sorting analysis showing increasing expression of canine CD34 after coculture with the murine cell line OP9. (G): Reverse-transcriptase PCR showing expression of CD34 and GATA2 mRNA after coculture (same cells as in [E, F]). Abbreviations: bp, base pairs; ES, canine ES-like cells; F, canine embryonic fibroblasts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, marker.

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To differentiate the canine embryo-derived stem cells into hematopoietic progenitor cells, a similar method as described for human ES cells by Kaufman et al. [11] was applied. Canine embryo-derived stem cells were cocultured with irradiated OP9 murine bone marrow stroma cells. At defined time points, the cells were harvested and subjected to fluorescence-activated cell sorting (FACS) analysis using an anti-canine CD34 monoclonal antibody or an isotype control antibody. Whereas only very few cells stained positive for CD34 at day 0, approximately 50% of the canine cells exhibited CD34 expression at days 6 and 9 (Fig. 1E, 1F). To further demonstrate differentiation toward the hematopoietic lineage, the mRNA expression of CD34 and GATA2, encoding a transcription factor specific for hematopoietic progenitor cells, was investigated by reverse transcriptase-PCR analysis (Fig. 1G). Expression of both genes was hardly detectable at day 0 but increased substantially after 6 and 9 days of coculture, consistent with the FACS data. To test the ability of the cells harvested from the cocultures to grow as colonies in colony-forming units (CFUs), the cells were cultured in 6-well plates. After 14 days, colonies were enumerated by light microscopy. Although cells from day 0 of coculture never did grow to colonies, cells from day 9 of coculture gave rise to CFU-M, CFU-E, CFU-G, and CFU-GM (0 colonies with cells from day 0 vs. 23 colonies with cells from day 9 of coculture) in one experiment. In another experiment, the responsiveness of the cells to hematopoietic growth factors was tested. Cells harvested from day 6 of coculture proliferated in response to a mixture of hematopoietic growth factors known to support the growth of canine hematopoietic progenitor cells [12], whereas much less proliferation was observed with cells harvested from day 0 of coculture (data not shown). Importantly, our in vitro differentiation results were obtained using cells at passages 10–12. Although the possibility of unlimited culture of these cells and their suitability for transplantation purposes remains to be demonstrated, we provide proof of principle of the practicability of this promising strategy.

Disclosures of Potential Conflicts of Interest

  1. Top of page
  2. Disclosures of Potential Conflicts of Interest
  3. Acknowledgements
  4. References
  5. Supporting Information

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Disclosures of Potential Conflicts of Interest
  3. Acknowledgements
  4. References
  5. Supporting Information

The authors thank the Else Kröner-Fresenius-Stiftung (Bad Homburg, Germany) for financial support. The excellent technical assistance by Steffen Schiller and Gisela Werner is also acknowledged. M.R.S. and H.A. contributed equally to this work.

References

  1. Top of page
  2. Disclosures of Potential Conflicts of Interest
  3. Acknowledgements
  4. References
  5. Supporting Information

Supporting Information

  1. Top of page
  2. Disclosures of Potential Conflicts of Interest
  3. Acknowledgements
  4. References
  5. Supporting Information
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Methods.pdf97KSupplemental Methods

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