Assembling Retinal Organoids with Microglia.

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

Effects of fluorescent protein tdTomato on mouse retina.

Fluorescent proteins (FPs) have been widely used to investigate cellular and molecular interactions and trace biological events in many applications. Some of the FPs have been demonstrated to cause undesirable cellular damage by light-induced ROS production in vivo or in vitro. However, it remains unknown if one of the most popular FPs, tdTomato, has similar effects in neuronal cells. In this study, we discovered that tdTomato expression led to unexpected retinal dysfunction and ultrastructural defects in the transgenic mouse retina. The retinal dysfunction mainly manifested in the reduced photopic electroretinogram (ERG) responses and decreased contrast sensitivity in visual acuity, caused by mitochondrial damages characterized with cellular redistribution, morphological modifications and molecular profiling alterations. Taken together, our findings for the first time demonstrated the retinal dysfunction and ultrastructural defects in the retinas of tdTomato-transgenic mice, calling for a more careful design and interpretation of experiments involved in FPs.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.

REG1A protects retinal photoreceptors from blue light damage.

With the increased use of artificial light and the prolonged use of optoelectronic products, light damage (LD) to the human retina has been identified as a global vision-threatening problem. While there is evidence of a significant correlation between light-induced retinal damage and age-related vision impairment in age-related macular degeneration, it is unclear how light-induced retinal degeneration manifests itself and whether there are agents capable of preventing the development of LD in the retina. This study investigated a mechanism by which blue light leads to photoreceptor death. By observing blue light exposure in retinal organoids and photoreceptor cells, we concluded that there could be significant apoptosis of the photoreceptors. We demonstrate that regenerating islet-derived 1 alpha (REG1A) prevents photoreceptors from undergoing this LD-induced apoptosis by increasing expression of the anti-apoptotic gene Bcl2 and downregulating expression of the pro-apoptotic gene Bax, resulting in reduced mitochondrial damage and improved aerobic capacity in photoreceptor cells. For the first time, REG1A has been shown to restore mitochondrial function and cell apoptosis after LD-induced damage, suggesting its potential application in the prevention and treatment of retinal vision loss.

Functional microglia derived from human pluripotent stem cells empower retinal organ.

Microglia are known to play essential roles in the development, progression and treatment of diverse neurodegenerative diseases in the central nervous system, including the retina, brain and spinal cord. Recently, brain-induced microglia-like cells (iMGs) have been generated from human pluripotent stem cells (hPSCs); however, retinal microglia have yet to be developed in vitro. In this study, by mimicking in vivo microglial development, we established a simplified approach to differentiate hPSCs into high purity (>90%) iMGs. The iMGs express microglia-specific markers, release cytokines upon stimulation, and are capable of phagocytizing bacteria. When co-cultured with three-dimensional human retinal organoids (hROs), iMGs migrated into the hROs, tended to differentiate into resident retinal microglia, and simultaneously induced apoptosis in some neural cells. Notably, the resident iMGs in the hROs formed sparse web-like structures beneath the photoreceptor cell layer, resembling microglia's orientation in human retina. In conclusion, we developed a simplified and efficient method to generate microglia from human pluripotent stem cells, and we report the first derivation of retinaresident microglia in vitro, providing a new source of human retinal microglia for developmental and disease studies and regenerative therapeutics.

Bioenergetic Crosstalk between Mesenchymal Stem Cells and various Ocular Cells through the intercellular trafficking of Mitochondria.

Rationale: Mitochondrial disorders preferentially affect tissues with high energy requirements, such as the retina and corneal endothelium, in human eyes. Mesenchymal stem cell (MSC)-based treatment has been demonstrated to be beneficial for ocular degeneration. However, aside from neuroprotective paracrine actions, the mechanisms underlying the beneficial effect of MSCs on retinal and corneal tissues are largely unknown. In this study, we investigated the fate and associated characteristics of mitochondria subjected to intercellular transfer from MSCs to ocular cells. Methods: MSCs were cocultured with corneal endothelial cells (CECs), 661W cells (a photoreceptor cell line) and ARPE-19 cells (a retinal pigment epithelium cell line). Immunofluorescence, fluorescence activated cell sorting and confocal microscopy imaging were employed to investigate the traits of intercellular mitochondrial transfer and the fate of transferred mitochondria. The oxygen consumption rate of recipient cells was measured to investigate the effect of intercellular mitochondrial transfer. Transcriptome analysis was performed to investigate the expression of metabolic genes in recipient cells with donated mitochondria. Results: Mitochondrial transport is a ubiquitous intercellular mechanism between MSCs and various ocular cells, including the corneal endothelium, retinal pigmented epithelium, and photoreceptors. Additionally, our results indicate that the donation process depends on F-actin-based tunneling nanotubes. Rotenone-pretreated cells that received mitochondria from MSCs displayed increased aerobic capacity and upregulation of mitochondrial genes. Furthermore, living imaging determined the ultimate fate of transferred mitochondria through either degradation by lysosomes or exocytosis as extracellular vesicles. Conclusions: For the first time, we determined the characteristics and fate of mitochondria undergoing intercellular transfer from MSCs to various ocular cells through F-actin-based tunneling nanotubes, helping to characterize MSC-based treatment for ocular tissue regeneration.

Prognostic factors on graft-versus-host disease-free and relapse-free survival after allogeneic hematopoietic stem cell transplantation for adults with acute leukemia.

The cure of acute leukemia by allogeneic hematopoietic stem cell transplantation (allo-HSCT) is closely linked to major complications leading to adverse outcomes, including graft-versus-host disease (GVHD), disease relapse and death. This study retrospectively investigated a consecutive series of 312 adult patients with acute leukemia receiving allo-HSCT by using a novel concept of GVHD-free/relapse-free survival (GRFS), and further evaluated the impact of clinical factors on GRFS. Results indicated that the 1- and 2-year GRFS were 54.8% and 51.5%, respectively. In multivariable analysis, recipient age >35years (HR 1.676; p=0.006), diagnosis of acute lymphoblastic leukemia (HR 1.653; p=0.027) and acute biphenotypic leukemia (HR 2.175; p=0.010), advanced disease (HR 2.702; p<0.001), and donor age >35 years (HR 1.622; p=0.008) were significantly associated with inferior GRFS post-HSCT. GRFS of haploidentical-related donor transplant was comparable to that of matched sibling donor or matched unrelated donor transplant. Furthermore, prophylactic donor lymphocyte infusion (DLI) had an overall beneficial effect on GRFS (HR 0.645, p=0.044). Collectively, with a better understanding of these significant prognostic factors which impacted on GRFS, we can effectively evaluate the risk and probability of real recovery after allo-HSCT, further optimizing the therapeutic avenues for acute leukemia.

Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of patients with mixed-lineage-leukemia-rearranged acute leukemia: Results from a prospective, multi-center study.

The purpose of this study is to define the role for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in mixed-lineage-leukemia (MLL)-rearranged acute leukemia, which is now poorly understood. A prospective, multi-center cohort study to determine whether allo-HSCT could decrease relapse rates and improve long-term survival of MLL+ leukemia patients was performed. Fifty-six consecutive patients diagnosed with MLL-rearranged acute leukemia undergoing allo-HSCT from two transplant centers in China were enrolled between October 2007 and October 2012. The trial was registered at www.chictr.org as # ChiCTR-ONC-12002739. The incidences of grades II to IV acute graft versus host disease (aGVHD) and of grades III and IV aGVHD were 28.8% (CI, 16.87-40.8%), and 14.2% (CI, 5.4-23.0%), respectively. The cumulative incidences for chronic GVHD (cGVHD) at 2 years after HSCT were 35.2% (CI, 21.2-49.2%). Up to April 30, 2013, 12 patients had relapsed and 11 died from relapse, and 37 patients were still alive without disease recurrence. The relapse and NRM rates at 3 years were 25.3% (CI, 12.7-37.9%) and 18.0% (CI, 2.6-33.4%), respectively. The probalities of overall survival and leukemia free survival were 61.8% (CI, 46.0-77.6%) and 56.3% (CI, 38.1-74.5%) at 3 years, respectively. Patients transplanted during their hematological first complete remission (CR1) had a lower relapse rate (17.9% vs. 48.1%, P = 0.03) compared with patients transplanted beyond CR1. The median overall survival for the 29 patients not receiving allo-HSCT during the study period was 145 days from diagnosis. This study showed that allo-HSCT could be a valuable treatment choice for MLL+ acute leukemia.