Comprehensive characterization of fetal and mature retinal cell identity to assess the fidelity of retinal organoids.

Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their in vivo counterpart. To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages.

Whole Genome Sequencing, Focused Assays and Functional Studies Increasing Understanding in Cryptic Inherited Retinal Dystrophies.

The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy.

Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic Variant Assessment for Therapy Suitability.

The RPGR gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in RPGR are the main contributor to X-linked rod-cone dystrophy (RCD), and RPGR gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel RPGR variants is important for a patient therapy opportunity.

Evaluation for Retinal Therapy for Variation Assessed in hiPSC Retinal Pigment Epithelial Cells.

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells.

Heterozygous COL9A3 variants cause severe peripheral vitreoretinal degeneration and retinal detachment.

The COL9A3 gene encodes one of the three alpha chains of Type IX collagen, with heterozygous variants reported to cause multiple epiphyseal dysplasia, and suggested as contributory in some cases of sensorineural hearing loss. Patients with homozygous variants have midface hypoplasia, myopia, sensorineural hearing loss, epiphyseal changes and carry a diagnosis of Stickler syndrome. Variants in COL9A3 have not previously been reported to cause vitreoretinal degeneration and/or retinal detachments.

Direct Comparison of Four Hematopoietic Differentiation Methods from Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are an invaluable resource for the study of human disease. However, there are no standardized methods for differentiation into hematopoietic cells, and there is a lack of robust, direct comparisons of different methodologies. In the current study we improved a feeder-free, serum-free method for generation of hematopoietic cells from iPSCs, and directly compared this with three other commonly used strategies with respect to efficiency, repeatability, hands-on time, and cost.

Pro-survival role of protein kinase C epsilon in Philadelphia chromosome positive acute leukemia.

Durable responses to imatinib monotherapy are rarely seen in aggressive forms of Philadelphia chromosome positive (Ph+) leukemias. To investigate the possible cause of treatment failure we examined the role of protein kinase C epsilon (PKCE), an oncogene highly implicated in the development of solid tumors and resistance to chemotherapy. We found high levels of PKCE transcripts in Ph+ acute lymphoblastic leukemia (ALL) cells from patients and cell lines, and imatinib resistant chronic myeloid leukemia, which were also less responsive to imatinib-induced apoptosis than Ph+ cells with lower PKCE expression.

Discriminating lymphomas and reactive lymphadenopathy in lymph node biopsies by gene expression profiling.

Background: Diagnostic accuracy of lymphoma, a heterogeneous cancer, is essential for patient management. Several ancillary tests including immunophenotyping, and sometimes cytogenetics and PCR are required to aid histological diagnosis. In this proof of principle study, gene expression microarray was evaluated as a single platform test in the differential diagnosis of common lymphoma subtypes and reactive lymphadenopathy (RL) in lymph node biopsies.

Two-step cross-entropy feature selection for microarrays—power through complementarity.

Current feature selection methods for supervised classification of tissue samples from microarray data generally fail to exploit complementary discriminatory power that can be found in sets of features. Using a feature selection method with the computational architecture of the cross-entropy method, including an additional preliminary step ensuring a lower bound on the number of times any feature is considered, we show when testing on a human lymph node data set that there are a significant number of genes that perform well when their complementary power is assessed, but “pass under the radar” of popular feature selection methods that only assess genes individually on a given classification tool. We also show that this phenomenon becomes more apparent as diagnostic specificity of the tissue samples analysed increases.

Lysosomal traffic of liganded endothelin B receptor.

The endothelin B receptor (ETB) is an endothelial cell receptor found in caveolae. Studies with GFP-tagged ETB have suggested that the protein is constitutively endocytosed and targeted to lysosomes where it is rapidly degraded. We report that iodinated endothelin-1 ligand (ET-1) is taken up by cells transfected with ETB and remains undegraded for at least 17 h.