We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses.

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, Visual System Homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium (RPE) at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wildtype VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans, and support the bona fide nature of hiPSC-OV-derived retinal progeny.

Epstein-Barr virus (EBV) establishes a latent form of infection in memory B cells, while antibody-secreting plasma cells often harbor the lytic form of infection. The switch between latent and lytic EBV infection is mediated by the two viral immediate-early proteins BZLF1 (Z) and BRLF1 (R), which are not expressed in latently infected B cells. Here we demonstrate that a cellular transcription factor that plays an essential role in plasma cell differentiation, X-box-binding protein 1 (XBP-1), also activates the transcription of the two EBV immediate-early gene promoters. In reporter gene assays, XBP-1 alone was sufficient to activate the R promoter, whereas the combination of XBP-1 and protein kinase D (PKD) was required for efficient activation of the Z promoter. Most importantly, the expression of XBP-1 and activated PKD was sufficient to induce lytic viral gene expression in EBV-positive nasopharyngeal carcinoma cells and lymphoblastoid cells, while an XBP-1 small interfering RNA inhibited constitutive lytic EBV gene expression in lymphoblastoid cells. These results suggest that the plasma cell differentiation factor XBP-1, in combination with activated PKD, can mediate the reactivation of EBV, thereby allowing the viral life cycle to be intimately linked to plasma cell differentiation. Epstein-Barr virus (EBV)is the causative agent of infectious mononucleosis and is associated with B-cell lymphomas, nasopharyngeal carcinomas, gastric carcinomas, and other malignancies (26,45). EBV causes lytic infection in normal oral epithelial cells (32, 51) while usually establishing one of the latent forms of infection in circulating memory B cells. In contrast, tonsillar B cells that express antigens specific for plasma markers commonly harbor the lytic form of EBV infection, which results in the production of infectious viral particles (10,29,30).The switch from latent to lytic EBV infection is mediated by the immediate-early (IE) protein BZLF1 (Z) and the immediate-early/early protein BRLF1 (R) (1,16,57). Z and R are transcription factors that activate each other's transcription and together are sufficient to activate the entire lytic viral gene expression cascade (17,49). In latently infected cells, the promoters driving Z and R expression (Zp and Rp) are inactive. Therefore, the activation of Zp and Rp by cellular transcription factors is the crucial initial step required for lytic viral gene expression. B-cell receptor engagement activates lytic EBV gene expression in some B-cell lines in vitro and activates both EBV IE promoters in reporter gene assays (23). Although several different individual cellular transcription factors can activate one or both of the two EBV IE promoters in reporter gene assays (23), to date these factors have not been shown to be sufficient for the efficient reactivation of lytic viral gene expression from the endogenous viral genome in latently infected cells.While there is a strong correlation between plasma cell differentiation and lytic EBV gene expression in human tonsils, it is not pr...

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations.We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hema- IntroductionPatient-specific induced pluripotent stem cells (iPSCs) can serve as useful models for understanding the etiology of disease and facilitating the development of novel therapeutic interventions. 1 B cells represent a larger fraction of the peripheral blood mononuclear cell population (ϳ 20%) and can be transformed in vitro by Epstein-Barr virus (EBV) to generate lymphoblastoid cell lines (LCLs) using as little as 0.5 mL blood, 2 creating an unlimited proliferative source of cells for reprogramming trials. LCLs are a precious resource for immunologic, epidemiologic, and rare disease studies. A number of facilities manage collections of LCLs available internationally to researchers. 2 Thus, generating iPSCs from LCLs offers the advantage of working with minimal amounts of blood from living donors as well as frozen LCL collections banked worldwide.The capability to reprogram terminally differentiated cells depends on the inherent physiologic plasticity of the cell type. B lymphocytes can transdifferentiate to macrophages 3,4 or hematopoietic precursor cells (HPCs) after down-regulation of Pax5 expression. 4 Murine B cells have been reprogrammed to iPSCs via viral transduction of reprogramming factors with 5 and without Pax5 inhibition. 6 Generating iPSCs via nonviral, nonintegrating methods is appealing to generate clinically useful iPSCs. Recently, iPSCs have been generated by delivering the reprogramming factors via oriP/EBNA-1-based plasmids in fibroblasts and peripheral blood CD34 ϩ cells. 7,8 The inherent plasticity of B cells, their receptivity to oriP/EBNA-1 plasmids, ease of generating LCLs, and availability of banked LCL collections inspired our efforts to reprogram LCLs using oriP/EBNA-1-based vectors.LCL-derived iPSCs (LCL-iPSCs) demonstrated the characteristics of pluripotent stem cells, a normal karyotype, the genetic identity, and IgGH signature of the parental LCLs and lost expression of the episomal reprogramming genes as well as viral genes, leading to self-sustained LCL-iPSCs essentially free of exogenous reprogramming and viral elements. MethodsDetailed methods are included in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). All animal experiments were conducted according to relevant national and international guideline...

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) mediates the switch between latent and lytic EBV infection. Z not only activates early lytic viral gene transcription but also plays a direct role in lytic viral genome replication. Although a small fraction of Z is known to be sumoylated, the effects of this posttranslational modification on various different Z functions have not been well defined. In this report, we show that only the lysine at amino acid residue 12 is required for the sumoylation of Z, and that Z can be sumoylated by SUMO isoforms 1, 2, and 3. We also demonstrate that the sumo-defective Z mutants ZK12A and ZK12R have enhanced transcriptional activity. The sumoylated and nonsumoylated forms of Z were found to have a similar cellular location, both being localized primarily within the nuclear matrix. The Z sumo-defective mutants were, however, partially defective for disrupting promyelocytic leukemia (PML) bodies compared to the ability of wild-type Z. In addition, we show that lytic viral genome replication does not require the sumoylation of Z, although a Z mutant altered at both amino acids 12 and 13 is replication defective. Furthermore, we show that the sumoylation of Z is greatly increased (from less than 1 to about 11%) in lytically induced 293 cells infected with an EBV mutant virus deleted for the EBV-encoded protein kinase (EBV-PK) compared to that of 293 cells infected with wild-type EBV, and that the overexpression of EBV-PK leads to the reduced sumoylation of Z in EBV-negative cells. Our results suggest that the sumoylation of Z helps to promote viral latency, and that EBV-PK inhibits Z sumoylation during viral reactivation.

The induction of lytic infection has been proposed as a therapeutic strategy for treating Epstein-Barr virus (EBV)-Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis, and EBV is associated with both epithelial and B-cell malignancies (reviewed in references 34 and 53). Like all herpesviruses, EBV can infect cells either latently or lytically. Lytic infection is required for the production of infectious viral particles, enabling the virus to spread from cell to cell and host to host. In the human host, lytic EBV infection is generally restricted to differentiated oropharyngeal epithelial cells and plasma cells. The switch from latent to lytic infection is mediated by the two viral immediate-early (IE) proteins, BZLF1 (also called Z, Zta, and ZEBRA) and BRLF1 (also called R and Rta). BZLF1 and BRLF1 are transcription factors that activate both each other's expression and, together, the entire lytic cascade of EBV gene expression (1,9,11,12,14,24,26,33,40,52,54,67). High-level expression of the BZLF1 gene or, in some cell lines, the BRLF1 gene is sufficient to convert cells from a latent to a lytic form of viral infection. In latently infected cells, the EBV IE genes are not transcribed. Therefore, the activation of one or both of these IE promoters by cellular transcription factors is the first crucial step in reactivation of EBV out of latency into its lytic cycle of replication.EBV infection of normal oropharyngeal epithelial cells in humans results in completely lytic infection (38; reviewed in references 34, 53, and 56). However, in EBV-associated epithelial malignancies, including nasopharyngeal and gastric carcinomas, most of the tumor cells contain one of the latent forms of viral infection (reviewed in references 29, 34, and 53). Presumably, the establishment of a predominantly latent form of EBV infection in these epithelial tumors helps both to ensure that the virus does not kill the tumor cells and to provide a selective growth advantage to the cells. Yet to be identified are the cellular and viral factors responsible for EBV infection of normal epithelial cells being completely lytic, yet converting to a latent form in epithelial tumor cells.EBV infection of most transformed human epithelial cell lines in vitro also usually leads to the establishment of cells containing EBV in a highly latent form. A notable exception to this general rule is the gastric carcinoma cell line AGS. AGS cells stably infected with the B95-8 strain of EBV support persistently lytic infection (32). Not yet understood is why, among transformed epithelial cell lines, AGS cells are uniquely susceptible to maintaining EBV infection in a highly lytic form.In this paper, we investigated viral and cellular factors that contribute to lytic EBV infection in AGS cells. We show that AGS cells stably infected with the B95-8 strain of EBV expressed a much higher level of viral lytic proteins than did stably infected HeLa cells, while expressing similar levels of EBNA-1, a latent protein. We examined the ac...

Lytically infected EBV-positive lymphoblastoid cells enhance the growth of early-passage, but not late-passage, EBV-immortalized lymphoblastoid cell lines (LCLs) in SCID mice and have enhanced IL-6 secretion. Here, we have examined the importance of IL-6 for the growth of early-passage LCLs (EPL) in SCID mice, identified lytic EBV proteins that activate IL-6 production and compared viral and cellular differences between early versus late passage LCLs (LPL). IL-6 was required for efficient growth of EPL in SCID mice. The EBV immediate-early (IE) proteins, BRLF1 and BZLF1, each induced IL-6 secretion when transfected into 293 and BJAB cells. Interestingly, the combination of BZLF1 and the latent EBV protein, LMP-1, induced much more IL-6 expression in both 293 and BJAB cells than either protein alone. Both BZLF1 and BRLF1 also enhanced IL-10 production in 293 cells. In comparison to the EPL, LPL had much reduced expression of early lytic viral proteins and cellular IL-6. In contrast, expression of cellular IL-10 was similar in EPL versus LPL, while VEGF secretion was increased in late-passage LCLs. These results suggest that both BRLF1 and BZLF1 contribute to IL-6 secretion in lytically infected cells and that lytically infected cells may promote early lymphoproliferative disease in patients through enhanced IL-6 production. ' 2007 Wiley-Liss, Inc.