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Piyathip Setthawong Praopilas Phakdeedindan Mongkol Techakumphu Theerawat Tharasanit 《Reproduction in domestic animals》2021,56(8):1104-1116
Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line. 相似文献
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Narong TIPTANAVATTANA Araya RADTANAKATIKANON Poul HYTTEL Hanne HOLM Supranee BURANAPRADITKUN Piyathip SETTHAWONG Mongkol TECHAKUMPHU Theerawat THARASANIT 《The Journal of reproduction and development》2015,61(6):581-588
The development of germ cells has not been entirely documented in the cat especially the transition phase of
the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development
and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes
were divided into 3 groups according to donor age (I, < 4 months; II, 4–6 months; and III, > 6 months).
In Exp. 1, we studied testicular development by histology, transmission electron microscopy and
immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow
cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3).
Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of
SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the
highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1+ cells (14.89 ±
5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation
in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed
mRNA for GFRA1, ZBTB16, RET and POU5F1.
Our study found that the G/SSC transition occurs at 4–6 months of age. This period is useful for isolation and
improves the establishment efficiency of cat SSCs in vitro. 相似文献
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