Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT

Abstract

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

“…The rates of SCNT blastocyst formation in bovine and mouse were more than 25% and were comparatively determined as higher efficiency than those of porcine SCNT (10%–17%) during in vitro pre‐implantation period. Furthermore, total cell number of porcine SCNT blastocysts consisted of 26–50 cells was generally lower than values from bovine or mouse SCNT blastocysts, which was more than 100 cells or approximately 60 cells, respectively (Chen et al., ; Costa‐Borges et al., ; Kumar et al., , ; Lee et al., ; Pan et al., ; Seaby et al., ). But in the present study, even after supplementing with autologous ooplasm into SCNT (AOT) embryos, no significant increases in the values of cleavage rate, blastocyst formation, hatched blastocyst and total cell number in comparison with SCNT embryos were observed.…”

“…It is generally addressed that the ratio of blastocyst formation in SCNT embryo during in vitro pre-implantation development is approximately 25%-33% in mouse and bovine, whereas that of porcine has still remained below 20% (Costa-Borges, Gonzalez, Santaló, & Ibáñez, 2011;Kumar et al, 2007;Lee et al, 2014;Pan et al, 2015;Seaby, Alexander, King, & Mastromonaco, 2013;Xu et al, 2013). As there have been considerable achievements of the application of OT, it can be possibly applied to porcine SCNT research to overcome low efficiency of embryo development.…”

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

“…The BFF were established from the bovine fetal back skin tissue of approximately 90-d-old Holstein cow fetuses (Pan et al, 2015). Briefly, fetal skin tissue samples were rinsed several times with PBS containing penicillin and streptomycin and then minced into 1-mm 3 pieces.…”

Procaine and S-adenosyl-L-homocysteine (SAH) affect the expression of genes related to the epigenetic machinery and change the DNA methylation status ofin vitrocultured bovine skin fibroblasts

“…Interestingly, we found that fibroblasts from all treatments expressed OCT4, even 448 fibroblasts from the control group, even though there were not expected to express 449 genes related to pluripotency because of their highly differentiated status. This result 450 may be explained by the results of Pan et al (2015) from their evaluation of skin 451 fibroblast samples showing the presence of a heterogeneous population of fibroblasts 452 containing multipotent stem cells (56). Moreover, we found no significant differences in 453 OCT4 transcript levels among the treatments, suggesting that the molecular changes 454 induced by procaine and SAH were not sufficient to alter the expression of pluripotency 455 genes such OCT4.…”