Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer.

AIM: To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene. METHODS: Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB) culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA. RESULTS: DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters. Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated. CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

Photoreceptor cells from mouse ES cells by co-culture with chick embryonic retina.

Degeneration of photoreceptors is a consistent and common endpoint in retinal diseases. Herein, we report the efficient induction of photoreceptor-like cells from mouse embryonic stem (ES) cells using chick embryonic retina tissue. Undifferentiated mouse ES cells were initially cultured in a preferential condition into a neural lineage, and ES cells were then co-cultured with chick embryonic day 6 (E6) retina tissues. After a 10-day co-culture, approximately 20% of the mouse ES derivatives became immuno-positive for rhodopsin. RT-PCR analysis demonstrated expression of the transcription factor crx and a distinct increase of rod photoreceptor-specific markers, IRBP and recoverin, after the start of the co-culture. These results indicate that co-culture of ES cells with chick embryonic retina tissue is a useful and efficient method for the induction of photoreceptor-like cells.

Expression of ciliary neurotrophic factor (CNTF), CNTF receptor alpha (CNTFR-alpha) following experimental intracerebral hemorrhage in rats.

Ciliary neurotrophic factor (CNTF) is known as a neuro-survival factor in the developing and developed CNS, as well as in the CNS following injury. However, little is known about the expression of CNTF or that of its receptor (CNTFR-alpha) in cases of intracerebral hemorrhage (ICH). We investigated the temporal and spatial profiles of CNTF and CNTFR-alpha expression using a collagenase-induced ICH rat model. CNTF expression was up-regulated from the day following ICH induction and reached a peak level at 5 to 14 days, with increased expression observed in brain tissue surrounding the hematoma lesion and white matter structures in association with astroglial proliferation. Further, CNTFR-alpha was transiently expressed in the cerebral cortex surrounding the hematoma, with a peak at 5 days. Administration of exogenous CNTF into the lesion following initiation of ICH resulted in a prolonged expression of CNTFR-alpha on cortical neurons neighboring the hematoma. Our findings suggest differential regulation of CNTF and CNTFR-alpha, and the possibility of a therapeutic strategy using CNTF administration for ICH.