Induced pluripotent stem (iPS) cells can be obtained from fibroblasts upon expression of Oct4, Sox2, Klf4, and c-Myc. To understand how these factors induce pluripotency, we carried out genome-wide analyses of their promoter binding and expression in iPS and partially reprogrammed cells. We find that target genes of the four factors strongly overlap in iPS and embryonic stem (ES) cells. In partially reprogrammed cells, many genes co-occupied by c-Myc and any of the other three factors already show an ES cell-like binding and expression pattern. In contrast, genes that are specifically co-bound by Oct4, Sox2, and Klf4 in ES cells and encode pluripotency regulators severely lack binding and transcriptional activation. Among the four factors, c-Myc promotes the most ES cell-like transcription pattern when expressed individually in fibroblasts. These data uncover temporal and separable contributions of the four factors during the reprogramming process and indicate that ectopic c-Myc predominantly acts before pluripotency regulators are activated.

Abstract SCI-41 The great excitement surrounding induced pluripotent stem (iPS) cells is tempered by the fact that little is known about how they are generated and why this process is so inefficient. We have set out to ask how the four transcription factors Oct4, Sox2, cMyc and Klf4 can induce pluripotency. By analyzing the role of the reprogramming factors Oct4, Sox2, Klf4 and cMyc during the initial step of reprogramming and comparing their binding and global expression in fully and partially reprogrammed cells, we previously uncovered that cMyc has a central role in the repression of the somatic cell expression program. With respect to the upregulation of the embryonic cell transcription, the four factors cooperate to activate the metabolic program, followed by binding of Oct4, Klf4 and Sox2 to genes encoding pluripotency regulators leading to the self-sustained, pluripotent state. Thus, we suggested that the factors have separable and temporal contributions to the reprogramming process. We are particularly interested in further deciphering the mechanism of reprogramming, understanding barriers of the reprogramming process, and comparing the embryonic stem cell state with the iPS cell state. Disclosures No relevant conflicts of interest to declare.

Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

BACKGROUND:Human rhinovirus (HRV) infections are associated with exacerbations of asthma, chronic obstructive pulmonary disease, and sinusitis. Nitric oxide (NO) might play an important role in host defense through its potent antiviral properties. Previous studies have shown that HRV infection in human subjects increased nasal epithelial expression of type 2 nitric oxide synthase (NOS2), an isoform of the enzyme that produces NO.OBJECTIVE:We sought to investigate whether increases in exhaled NO (eNO) would accompany the increased NOS2 expression and would be associated with clearance of the virus.METHODS:Six human subjects were infected with HRV-16 intranasally. eNO from nasal and lower airways was measured by means of direct measurement at multiple controlled flow rates. eNO was monitored at baseline (day 1) and on days 2 to 5, 8, 14, and 42 after infection. Nasal lavages were performed on days 1 to 5 and 8, and nasal scrapings were performed on days 1 to 4. NOS2 mRNA expression in nasal cells was measured by using quantitative real-time RT-PCR. Viral shedding in nasal lavage fluid was monitored by using real-time RT-PCR and bioassay.RESULTS:Peak HRV titers and symptom scores were correlated on day 3, and HRV persisted until day 5 (n=4) or day 8 (n=2). Infection was associated with transient but significant increases in lymphocytes and monocytes in nasal lavage fluid. Significant increases in both nasal and lower airway eNO concentrations accompanied HRV infection and were positively correlated. Increased nasal eNO concentrations on day 3 were associated with increased expression of NOS2 mRNA in nasal scrapings. Symptom scores on day 4 were inversely correlated with the increases in nasal eNO concentration.CONCLUSIONS:We conclude that increased production of NO occurs as part of the host response to HRV infection and speculate that NO plays a beneficial role in viral clearance.