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...