Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny1

Abstract

The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here, we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts, or adipocytes. Adipocytes upregulated adipogenic markers procollagen type I alpha 2 (COL1A2), peroxisome proliferator-activated receptor gamma 2 (PPARG), and gylceraldehyde-3-phosphate dehydrogenase (GAPDH), and downregulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor-specific markers COL1A2, PPARG, GAPDH, and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61, and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone, and fat cells), had no effect on in vitro development to blastocysts (93 [38%] of 248 vs. 32 [44%] of 73 vs. 59 [32%] of 183, respectively). Likewise, development to weaning was not significantly different between the three cell types (2 [4%] of 46 vs. 2 [29%] of 7 vs. 4 [13%] of 31, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes

“…The higher appropriate remodeling of stem cell-NT derived embryos has proved in different species (Bosch et al, 2006, Faast et al, 2006, Kumar et al, 2012, Lee et al, 2006, Rideout et al, 2000. Some reports, however, have rejected transcendence of stem cells over somatic cells in normal reprogramming of NT derived embryos (Berg et al, 2007, Li et al, 2013, Sung et al, 2006. In the first report on the use of bovine MSCs for NT, no priority in developmental potential was observed in stem cell derived embryos compared to non-stem cell derived counterparts (Kato et al, 2004).…”

Deer antler – A novel model for studying organ regeneration in mammals

“…Clearly, both populations of cells can give rise to chondrocytes (Fig. 6A) and osteoblasts, respectively, when in micromass culture (Li and Suttie, 2006) and in media containing dexamethasone and ascorbate (Berg et al, 2007). As both the pedicles and antlers are composed of cartilage and bone, it was not surprising to learn that the AP and the PP cells were able to differentiate into chondroblastic and osteoblastic lineages (Kierdorf et al, 1994;Li and Suttie, 1994).…”

“…The differentiation potency of the AP and the PP cells has been investigated by several laboratories (Berg et al, 2007;Li and Suttie, 2006;Price et al, 2005). Clearly, both populations of cells can give rise to chondrocytes (Fig.…”

Deer Antlers – A Model of Mammalian Appendage Regeneration: An Extensive Review

“…It has been suggested that the (cambium layer of the) AP contains stem cells and that pedicle and first antler growth depend on the activation of these stem cells [14,15] . It was shown that cells from the AP express the transcription factor gene OCT-4 (POU5F1) and that the expression of this gene is downregulated in cultured AP cells upon differentiation into adipocytes [29] . More recently it was reported that cells from the AP also express considerable levels of the cell surface antigen CD9 [15] .…”

“…More recently it was reported that cells from the AP also express considerable levels of the cell surface antigen CD9 [15] . Cells isolated from the AP of red deer (Cervus elaphus) could be induced to differentiate along different (osteoblastic and adipogenic) mesenchymal lineages in vitro [29] . An in vivo study applying AP cell labeling with the lacZ gene at the time of pedicle initiation observed ␤ -galactosidaseexpressing cells in mesenchyme, precartilage, cartilage, and bone (but not skin) of the growing appendage, thereby confirming the derivation of pedicles and first antlers from the AP [15] .…”