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    • Chloroquine Epiretinal transplantation does not involve reti

    2018-10-24

    Epiretinal transplantation does not involve retinal detachment and may cause less trauma to the retina as compared with subretinal cell delivery. However vitreous detachment in patients causes increased photopsias, floaters and increased risk of retinal detachment and macular holes (de Smet et al., 2013; Witmer and Cohen, 2013). In future studies we will examine the safety of this procedure in large animal models that closely mimic the human eye. The following are the supplementary data related to this article.
    Introduction Induced hematopoietic stem Chloroquine (iHSCs) derived from either pluripotent stem cells (PSCs) or somatic cells are essential to cell therapy based on HSC transplantation. However, the derivation of iHSCs with physiologic functions after engraftment has remained a significant challenge (Slukvin, 2013). Several bottlenecks, including the key conditions of hematopoietic specification, maturation, maintenance, and expansion, must be addressed (Shepard and Talib, 2014). The ectopic expression of intrinsic regulatory factors has been hypothesized to directly convert somatic cell types to hematopoietic stem cells. As a result, HSC-like cells have been obtained via the conversion of pro-B (Riddell et al., 2014) and PSC-derived hematopoietic progenitors (Doulatov et al., 2013). In addition to some defects, these iHSCs may constitute a tumorigenic risk due to the enforced expression of factors via lenti- or retro-viral transduction approaches. To date, the in vitro niche modeling of hematopoietic development has failed to yield functional HSCs from PSCs (Kennedy et al., 2012), indicating that current extrinsic niche factors are not sufficient for the derivation of iHSCs from PSCs in vitro without the direct combination of manipulating intrinsic factors. Recently, iHSCs were identified to result from intra-teratoma hematopoiesis with low efficiency (Amabile et al., 2013; Suzuki et al., 2013). The efficiency of iHSCs derived from teratomas was further increased by combining the OP9- and OP9-derived-cell lines as well as hematopoietic cytokines. Thus, teratoma hematopoiesis constitutes a new 3-D in vivo model for studying niche factors for the derivation of iHSCs from PSCs. Lhx2 is a LIM homeobox gene that plays important roles in eye, forebrain, and definitive erythrocyte development. Knocking out Lhx2 leads to severe anemia in a cell non-autonomous manner (Porter et al., 1997). The enforced expression of Lhx2 in hematopoietic progenitors from either PSCs (Pinto do et al., 1998) or adult mouse bone marrow (Pinto do et al., 2002) results in SCF-dependent cell lines via a cell non-autonomous mechanism (Pinto do et al., 2001). PSCs that overexpress Lhx2 can produce HSC-like cells via OP9/PSCs co-culture; however, the related mechanism remains elusive (Kitajima et al., 2011). In addition to Lhx2, multiple transcription factors, including Fos, Tcfec, Hmgb1, and Sfpi1, can enhance the repopulating activity of HSCs in a cell non-autonomous manner (Deneault et al., 2009). We hypothesize that certain transcription factors can enhance the hematopoietic specification of PSCs in a cell non-autonomous manner. We report for the first time that the enforced expression of Lhx2 in OP9 stromal cells promotes the production of hematopoietic progenitors both in vitro and in vivo. OP9-Lhx2 facilitates hematopoietic specification partially via the upregulation of the Apln gene. Thus, the OP9-Lhx2 cell line can be widely used for hematopoietic derivation from PSCs, both in vitro and in vivo.
    Materials and methods
    Results
    Discussion The generation of induced HSCs from PSCs is an ideal approach for overcoming the lack of suitable HSC resources and graft rejection. Attempts to derive robustly induced HSCs from PSCs in recent decades failed due to multiple bottlenecks, including limited approaches for identifying and manipulating the stemness of HSCs in vitro (Shepard and Talib, 2014). In this study, we established a niche cell line, OP9-Lhx2 that can promote hematopoietic derivation using in vitro co-culture and in vivo teratoma formation methods. Interestingly, the ability of OP9-Lhx2 to facilitate the production of hematopoietic progenitors from PSCs is conserved from murine to human PSCs. Previous modifications of OP9 cells directly focused on extrinsic factors that play roles in hematopoietic development (Holmes and Zuniga-Pflucker, 2009; Ichii et al., 2012). In this study, we developed a new approach and showed that the ectopic expression of transcription factors in OP9 cells can also improve the niche to produce hematopoietic progenitors from PSCs. A mechanistic analysis demonstrated that OP9-Lhx2 promotes hematopoietic derivation partially via the upregulation of Apln, which has been reported to enhance the hematopoietic specification of PSCs in vitro (Yu et al., 2012). Because Lhx2 regulated the expression level of several genes involved in cell adhesion/signaling pathways, additional effectors might also enhance the hematopoietic specification by OP9-Lhx2.