Rapid Loss of Oct-4 and Pluripotency in Cultured Rodent Blastocysts and Derivative Cell Lines1

Abstract

The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.

Isolation and Characterization of Kidney-Derived Stem Cells

“…MRPC were isolated from adult rat kidneys using culture conditions that were similar to those used for the culture of bone marrow-derived multipotent adult progenitor cells with the exception that we did not deplete the cells that were positive for CD45 or glycophorin A (27). The source for the rat kidneys were 2-to 4-mo-old Fisher rats, including Oct4 ␤-Geo transgenic rats that contain a transgene that combines a neomycinresistance gene with a lacZ reporter under the control of 3.6 kb of the mouse Oct4 upstream sequence, including both proximal and distal enhancers (gift from Dr. Austin Smith, University of Edinburgh, Edinburgh, Scotland) (28). Oct4 is a POU family transcription factor that is expressed in embryonic and adult stem cells and immortalized nontumorigenic cell lines and tumor cells but not in differentiated cells (27,29 -31).…”

The Potential of Rat Inner Cell Mass and Fetal Neural Stem Cells to Generate Chimeras

“…These results supported the earlier report, in which Ouhibi et al (1995) indicated that rat ICM cells differentiated rapidly after 3-6 days in primary culture even when they were cultured on fibroblast feeder layers. Potential of cultured ICM cells decrease rapidly due to the loss of POU-domain transcription factor Oct-4 expression following culture in vitro, because expression of Oct-4 is essential for cells to maintain their pluripotency (Buehr et al, 2003). Rat chimera had not been gengerated in this study, it might be the same reason, but it still needs to be investigated.…”

“…So far, there have only been reports demonstrating the production of rat chimeras by aggregation of 8-cell embryos or morulae (Weinberg et al, 1985;Clegg et al, 1991), or by 8-cell embryos injected with ICMs (Buehr et al, 2003). Here we have generated rat chimeras by SD or DA ICM exchange injection between SD and DA rat strains.…”

“…Rat chimera had not been gengerated in this study, it might be the same reason, but it still needs to be investigated. These results suggest that rat ICM cells are very different from mouse ICM cells because germline chimeric mice can be efficiently produced by mouse ES cells that were even cultured in the medium with synthetic serum and in the absence of a feeder layer for a long period time (Buehr et al, 2003). To Maintain the potential of rat ICM cells in vitro for gene modification therefore remains a challenge.…”

“…The first rat chimera from ES cells was reported in 1994, but the chimera actually was rat-mouse interspecies chimera because the rat ES cells were accidentally contaminated with mouse ES cells (Iannaccone et al, 1994;Brenin et al, 1997). So far, rat chimeras have been generated by aggregation of rat 8-cell embryos or morulae (Weinberg et al, 1985;Clegg et al, 1991) and by injection of freshly isolated inner cell mass (ICM) cells of blastocysts into 8-cell embryos (Buehr et al 2003). Except for these limited studies described above, there have not been reports about production of rat chimeras from blastocyst injection with ICM cells or ES cells.…”