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  • In this study we transplant

    2018-11-09

    In this study, we transplant hESC-RPE to a large-eyed animal. Human and rabbit eyes are of similar size, the main advantage compared with previously studied mouse and rat models (Carido et al., 2014; Lund et al., 2006). Transplantation in rodents requires high cell concentrations (i.e., typically 50,000 cells/μl or more) and a transscleral approach through the choroid (Vugler et al., 2008). The outer blood-retinal barrier is thus compromised, potentially triggering an inflammatory response. Moreover, high cell concentrations and limited surgical control may cause misdirection, multilayering, and clumping of transplanted cells. Indeed, several publications have shown that photoreceptor rescue is neither RPE specific nor correlated with an intact donor cell layer (eg. Pinilla et al., 2009). The large-eyed rabbit allowed us to perform a surgical technique with instrumentation identical to a clinical setting (el Dirini et al., 1992). The model also permitted high-resolution in vivo tracking of transplanted phospholipase c inhibitor and monitoring of the overlying neurosensory retina through time. Using this methodology, we demonstrated subretinal monolayers of rhLN-521-hESC-RPE that remained for up to 8 months. Furthermore, integrated cells possessed in vivo functionality including phagocytic activity and rescue of photoreceptors from induced degeneration. Importantly, this effect showed both specificity and sensitivity as non-RPE and non-integrated RPE cells were ineffective. Suspension transplants have been frequently used in rodent models (Carido et al., 2014; Idelson et al., 2009; Lund et al., 2006; Vugler et al., 2008) and ongoing clinical studies (Schwartz et al., 2012, 2015). Concerns about this delivery method were raised with the main criticism being possible multilayering of donor cells leading to inefficient integration (Schwartz et al., 2012). As an alternative, transplantation of cells as prepolarized sheets with or without a supporting biomatrix has been suggested (Diniz et al., 2013; Stanzel et al., 2014). However, these large transplants are surgically demanding and may lead to retinal scarring and outer retinal degeneration (Kamao et al., 2014; Stanzel et al., 2014). In the present study, we noted minimal retinal scarring and a well-preserved neurosensory retina overlying the transplanted monolayer. Moreover, by using suspension transplants, we obtained monolayers up to ten times the size of RPE sheets with a typical size of 2–2.5 mm2 (Kamao et al., 2014; Stanzel et al., 2014). We demonstrate that a minimally invasive surgical procedure in a large-eyed disease model can achieve high-yield functional long-term hESC-RPE integration with photoreceptor preservation. These findings have important implications for ongoing and future clinical studies for the development of a safe and efficient cell replacement therapy for GA.
    Experimental Procedures
    Author Contributions
    Acknowledgments This work was supported by grants from the Swedish Research Council (VR), Ragnar Söderberg Foundation, Swedish Foundation for Strategic Research, Stockholm County Council (ALF project), Ögonfonden and Cronqvist Foundation. This study was performed at the WIRM flow cytometry facility, supported by Knut och Alice Wallenbergs Stiftelse (KAW) and the Live Cell Imaging Unit/Nikon Center of Excellence, supported by KAW, VR, Centre for Innovative Medicine, and the Jonasson donation.
    Introduction Proximal airway epithelial cells (PAECs) play a pivotal role in the host defense in the respiratory tract via mucociliary clearance organized by multi-ciliated airway cells (MCACs) and secretory cells. An abnormal function of MCACs is associated with various lung diseases such as primary ciliary dyskinesia (PCD) (Rossman et al., 1980) and cystic fibrosis (CF) (Zhang et al., 2009). It has been reported that PAECs could be generated from human pluripotent stem cells (hPSCs) involving human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Mou et al., 2012; Wong et al., 2012; Huang et al., 2014; Firth et al., 2014). The ciliary movement of hPSC-derived MCACs has not yet been reported, although that of murine embryonic stem cell-derived MCACs has been reported (Nishimura et al., 2006; Shojaie et al., 2015). In our previous study, we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs) and demonstrated the potency of CPM+ VAFECs to differentiate into alveolar type II cells (Gotoh et al., 2014). We hypothesized that PAECs could also be induced from CPM+ VAFECs, as all lung epithelial lineage cells have been reported to be differentiated from NKX2-1+ VAFECs (Kimura et al., 1996). We herein report a method of directed differentiation of hPSCs into MCACs and pulmonary neuroendocrine cells (PNECs) and functional analyses of the ciliary movement of hPSC-derived MCACs.