Additional file 1 of Establishment of a human pluripotent stem cell-derived MKX-td Tomato reporter system

Additional file 1. Supplemental Fig. S1 Full-lenght blot related to Fig. 1c

Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming

<p>Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous <i>cis</i>-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.</p

Three-Component Coupling Based on the “Cation Pool” Method

Sequential one-pot three-component coupling reactions have been developed based on the “cation pool” method. An N-acyliminium ion generated by the “cation pool” method adds to an electron-rich carbon−carbon double bond, such as enamine derivatives and vinyl sulfides, to form the second “cation pool”. The addition of nucleophiles such as allylsilanes, enol silyl ethers, Grignard reagents, and organoaluminum compounds led to the formation of the corresponding three-component coupling products

The number of c-Fos positive cells in PVN after administration of CNO.

Immunohistochemical staining of c-Fos in the PVN 120 min after an intraperitoneal CNO administration (1.0 mg/kg). An overview of the experimental schedule is presented in (A). Image of PVN in saline-treated mice (B) and CNO-treated mice (C). The number of c-Fos positive cells in the saline and CNO (D). Data represents mean ± SEM (n = 4–5). Statistical analyzes were performed as unpaired t-tests. **P < 0.01.</p

Effects of activation of OXTergic neurons in PVN on the Y-maze test and NORT.

Mice received an intraperitoneal administration of saline or CNO (1 mg/kg) 20 min before Y-maze. An overview of the experimental schedule is presented in (A). Spontaneous alternation (B). Total arm entries (C). Mice received an intraperitoneal administration of saline or CNO (1 mg/kg) 120 min before the training session in NORT. An overview of the experimental schedule is presented in (D). Exploration time of object (%) during the training session (E). Exploration time of novel object (%) during the test session (F). Data were presented as the mean ± SEM (B, C; n = 7–8: E, F; n = 11–12). Statistical analyzes were performed as unpaired t-tests. ***P < 0.001, ns: no-significant.</p

S4 Raw data -

Oxytocin (OXT) neurons project to various brain regions and its receptor expression is widely distributed. Although it has been reported that OXT administration affects cognitive function, it is unclear how endogenous OXT plays roles in cognitive function. The present study examined the role of endogenous OXT in mice cognitive function. OXT neurons were specifically activated by OXT neuron-specific excitatory Designer Receptors Exclusively Activated by Designer Drug expression system and following administration of clozapine-N-oxide (CNO). Object recognition memory was assessed with the novel object recognition task (NORT). Moreover, we observed the expression of c-Fos via immunohistochemical staining to confirm neuronal activity. In NORT, the novel object exploration time percentage significantly increased in CNO-treated mice. CNO-treated mice showed a significant increase in the number of c-Fos-positive cells in the supramammillary nucleus (SuM). In addition, we found that the OXT-positive fibers from paraventricular hypothalamic nucleus (PVN) were identified in the SuM. Furthermore, mice injected locally with CNO into the SuM to activate OXTergic axons projecting from the PVN to the SuM showed significantly increased percentage time of novel object exploration. Taken together, we proposed that object recognition memory in mice could be modulated by OXT neurons in the PVN projecting to the SuM.</div

S3 Raw data -

Oxytocin (OXT) neurons project to various brain regions and its receptor expression is widely distributed. Although it has been reported that OXT administration affects cognitive function, it is unclear how endogenous OXT plays roles in cognitive function. The present study examined the role of endogenous OXT in mice cognitive function. OXT neurons were specifically activated by OXT neuron-specific excitatory Designer Receptors Exclusively Activated by Designer Drug expression system and following administration of clozapine-N-oxide (CNO). Object recognition memory was assessed with the novel object recognition task (NORT). Moreover, we observed the expression of c-Fos via immunohistochemical staining to confirm neuronal activity. In NORT, the novel object exploration time percentage significantly increased in CNO-treated mice. CNO-treated mice showed a significant increase in the number of c-Fos-positive cells in the supramammillary nucleus (SuM). In addition, we found that the OXT-positive fibers from paraventricular hypothalamic nucleus (PVN) were identified in the SuM. Furthermore, mice injected locally with CNO into the SuM to activate OXTergic axons projecting from the PVN to the SuM showed significantly increased percentage time of novel object exploration. Taken together, we proposed that object recognition memory in mice could be modulated by OXT neurons in the PVN projecting to the SuM.</div

S1 Raw data -

Oxytocin (OXT) neurons project to various brain regions and its receptor expression is widely distributed. Although it has been reported that OXT administration affects cognitive function, it is unclear how endogenous OXT plays roles in cognitive function. The present study examined the role of endogenous OXT in mice cognitive function. OXT neurons were specifically activated by OXT neuron-specific excitatory Designer Receptors Exclusively Activated by Designer Drug expression system and following administration of clozapine-N-oxide (CNO). Object recognition memory was assessed with the novel object recognition task (NORT). Moreover, we observed the expression of c-Fos via immunohistochemical staining to confirm neuronal activity. In NORT, the novel object exploration time percentage significantly increased in CNO-treated mice. CNO-treated mice showed a significant increase in the number of c-Fos-positive cells in the supramammillary nucleus (SuM). In addition, we found that the OXT-positive fibers from paraventricular hypothalamic nucleus (PVN) were identified in the SuM. Furthermore, mice injected locally with CNO into the SuM to activate OXTergic axons projecting from the PVN to the SuM showed significantly increased percentage time of novel object exploration. Taken together, we proposed that object recognition memory in mice could be modulated by OXT neurons in the PVN projecting to the SuM.</div

Expressions of c-Fos in the various brain areas after NORT.

Immunohistochemical staining of c-Fos 120 min after the NORT. Table showed the number of c-Fos positive cells in the PRh, Ent, CA1, CA2, CA3 and Cg/RS. Data were presented as the mean ± SEM. Statistical analyzes were performed as unpaired t-tests.</p

Effects of activation of OXTergic neurons in SuM during the NORT.

Mice received a local injection of saline or CNO (3μM, 200nL) to SuM 20 min before the training session in NORT. An overview of the experimental schedule is presented in (A). Exploration time of object (%) during the training session (B). Exploration time of novel object (%) during the test session (C). Data were presented as the mean ± SEM (B, C; n = 5). Statistical analyzes were performed as unpaired t-tests. *P < 0.05, ns: no-significant.</p