Stem cell (SC) research has been around for nearly 40 years, but only human and mouse SCs have been characterized, have consensus protocols and standardized procedures. For other animals, including domestic ones, this is not the case. There is no established methodology and big efforts are being made by the scientific community in this direction, our lab included. Pluripotent stem cells (PSCs) have the capacity to self-renew indefinitely and to develop into the three primary germ cell layers and therefore to form all lineages of the body.
PSCs can be derived using two parallel approaches: (1) the Inner Cell Mass (ICM) of a bovine blastocyst and (2) reprogramming somatic cells to pluripotent state by ectopic expression of various pluripotency factors -e.g. induced pluripotent stem cells (iPS). Currently, our research focuses on producing iPS from bovine fetal fibroblasts (BFF). In this process, the fibroblasts, which are somatic cells, undergo reprogramming to PSCs.
Bovine fetuses of Holstein cows were obtained from a local abattoir. The fibroblastic tissue was processed under sterile conditions and either directly plated with growth media or digested using a mix of 4 enzymes to obtain the fibroblast cells and plate with the media. The cells were grown for 2 passages and then infected with 7 lentivirus vectors for pluripotent genes. The vectors were then induced with Doxycycline. Four weeks later, the colonies were isolated and grown separately with appropriate growth conditions for stem cells. RNA was extracted from the colonies at passages 1-2 and qRT-PCR was performed to assess pluripotency. Additionally, IF was performed.
We screened for 11 medias that can support infected fibroblasts reprograming to iPS, and identified 4 potential medias for further examination. Using these four medias, we managed to culture partly pluripotent cells.
Bovine fibroblast reprograming:
A: RT-PCR results for 3 pluripotent markers. Graph columns: C - different medias, ICM (inner cell mass) – positive control, BFF (bovine fetus fibroblasts) - negative controls .B: IF for 4c media using different antibodies. DAPI labels DNA. Images were taken in confocal microscope (x63, Zeiss).
Next, we will use the knowledge and skills gained toward the generation of true pluripotent embryonic stem cells from bovine and other domesticated animals. We plan to examine the molecular and epigenetic characteristics of these bovine ESC and compare them to human and mouse ESCs.
Embryonic stem cell-based approach is expected to have many applications in domestic animals such as dogs, pigs, goats and cows. Large animals are considered excellent models for long-term experiments in regenerative medicines and biomedical research in general, because of their similarities in physiology with humans as contrasted with the laboratory mouse or rat. Thus, establishing of naïve embryonic stem cell lines from domesticated animals can benefit both clinical applications to improve human health and agricultural applications.