Generation of four induced pluripotent stem cells lines from PBMC of the DFNA58 family members: Two hearing-impaired duplication carriers (USPi006-A e USPi007-A) and two normal-hearing noncarriers (USPi004-A and USPi005-A).

The DFNA58 locus contains a genomic duplication involving three protein-coding genes (CNRIP1, PLEK, and PPP3R1's exon 1) and other uncharacterized lncRNA genes (LOC101927723, LOC107985892 and LOC102724389). To clarify the role of these genes in hearing and precisely determine their role in hearing loss, four iPSC lines were generated from two carriers and two noncarriers of the duplication.

Single retinal image for diabetic retinopathy screening: performance of a handheld device with embedded artificial intelligence.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness. Our objective was to evaluate the performance of an artificial intelligence (AI) system integrated into a handheld smartphone-based retinal camera for DR screening using a single retinal image per eye. METHODS: Images were obtained from individuals with diabetes during a mass screening program for DR in Blumenau, Southern Brazil, conducted by trained operators. Automatic analysis was conducted using an AI system (EyerMaps™, Phelcom Technologies LLC, Boston, USA) with one macula-centered, 45-degree field of view retinal image per eye. The results were compared to the assessment by a retinal specialist, considered as the ground truth, using two images per eye. Patients with ungradable images were excluded from the analysis. RESULTS: A total of 686 individuals (average age 59.2 ± 13.3 years, 56.7% women, diabetes duration 12.1 ± 9.4 years) were included in the analysis. The rates of insulin use, daily glycemic monitoring, and systemic hypertension treatment were 68.4%, 70.2%, and 70.2%, respectively. Although 97.3% of patients were aware of the risk of blindness associated with diabetes, more than half of them underwent their first retinal examination during the event. The majority (82.5%) relied exclusively on the public health system. Approximately 43.4% of individuals were either illiterate or had not completed elementary school. DR classification based on the ground truth was as follows: absent or nonproliferative mild DR 86.9%, more than mild (mtm) DR 13.1%. The AI system achieved sensitivity, specificity, positive predictive value, and negative predictive value percentages (95% CI) for mtmDR as follows: 93.6% (87.8-97.2), 71.7% (67.8-75.4), 42.7% (39.3-46.2), and 98.0% (96.2-98.9), respectively. The area under the ROC curve was 86.4%. CONCLUSION: The portable retinal camera combined with AI demonstrated high sensitivity for DR screening using only one image per eye, offering a simpler protocol compared to the traditional approach of two images per eye. Simplifying the DR screening process could enhance adherence rates and overall program coverage.

TRANSPLANTATION OF SUBRETINAL STEM CELL-DERIVED RETINAL PIGMENT EPITHELIUM FOR STARGARDT DISEASE: A Phase I Clinical Trial.

PURPOSE: To assess the safety of injecting human embryonic stem cell retinal pigment epithelial cell dose to treat Stargardt disease. METHODS: In this prospective, Phase I clinical trial, human embryonic stem cell retinal pigment epithelial cells in suspension were injected into the subretinal space in eyes with the worse best-corrected visual acuity (BCVA). After vitrectomy/posterior hyaloid removal, a partial retinal detachment was created and the human embryonic stem cell retinal pigment epithelial cells were administered. Phacoemulsification with intraocular lens implantation was performed in eyes with lens opacity. All procedures were optical coherence tomography-guided. The 12-month follow-up included retinal imaging, optical coherence tomography, visual field/electrophysiologic testing, and systemic evaluation. The main outcome was the absence of ocular/systemic inflammation or rejection, tumor formation, or toxicity during follow-up. RESULTS: The mean baseline BCVAs in the phacoemulsification and no phacoemulsification groups were similar (1.950 ± 0.446 and 1.575 ± 0.303, respectively). One year postoperatively, treated eyes showed a nonsignificant increase in BCVA. No adverse effects occurred during follow-up. Intraoperative optical coherence tomography was important for guiding all procedures. CONCLUSION: This surgical procedure was feasible and safe without cellular migration, rejection, inflammation, or development of ocular or systemic tumors during follow-up.

Kidney organoids generated from erythroid progenitors cells of patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis. METHODS: Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. RESULTS: iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. CONCLUSION: The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD. METHODS: We transplanted mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPC and hiPSC cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology, and immunohistochemistry were evaluated 45 days post-surgery. RESULTS: We successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups. CONCLUSIONS: In conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seem to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improves clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.

In vitro differentiation of cGMP-grade retinal pigmented epithelium from human embryonic stem cells.

BACKGROUND: The World Health Organization (WHO) estimates that the number of individuals who lose their vision due to retinal degeneration is expected to reach 6 million annually in 2020. The retinal degenerative diseases affect the macula, which is responsible for central and detailed vision. Most macular degeneration, i.e., age-related macular degeneration (AMD) develops in the elderly; however, certain hereditary diseases, such as the Stargardt disease, also affect young people. This degeneration begins with loss of retinal pigmented epithelium (RPE) due to formation of drusen (atrophic) or abnormal vessels (exudative). In wet AMD, numerous drugs are available to successful treat the disease; however, no proven therapy currently is available to treat dry AMD or Stargardt. Since its discovery, human embryonic stem cells (hESCs) have been considered a valuable therapeutic tool. Some evidence has shown that transplantation of RPEs differentiated from hESCs cells can result in recovery of both RPE and photoreceptors and prevent visual loss. METHODS: The human embryonic WA-09 stem cell lineage was cultured under current Good Manufacturing Practices (cGMP) conditions using serum-free media and supplements. The colonies were isolated manually and allowed to spontaneously differentiate into RPE cells. RESULTS: This simple and effective protocol required minimal manipulation and yielded more than 10e8 RPE cells by the end of the differentiation and enrichment processes, with cells exhibiting a cobblestone morphology and displaying cellular markers and a gene expression profile typical of mature RPE cells. Moreover, the differentiated cells displayed phagocytic activity and only a small percentage of the total cells remained positive for the Octamer-binding transcriptions factor 4 (OCT-4) pluripotency cell marker. CONCLUSIONS: These results showed that functional RPE cells can be produced efficiently and suggested the possibility of scaling-up to aim at therapeutic protocols for retinal diseases associated with RPE degeneration.

Induced Pluripotent Stem Cells Reduce Progression of Experimental Chronic Kidney Disease but Develop Wilms' Tumors.

The therapeutic effect of induced pluripotent stem cells (iPSs) on the progression of chronic kidney disease (CKD) has not yet been demonstrated. In this study, we sought to assess whether treatment with iPSs retards progression of CKD when compared with bone marrow mesenchymal stem cells (BMSCs). Untreated 5/6 nephrectomized rats were compared with CKD animals receiving BMSCs or iPSs. Renal function, histology, immunohistochemistry, and gene expression were studied. Implanted iPSs were tracked by the SRY gene expression analysis. Both treatments minimized elevation in serum creatinine, significantly improved clearance, and slowed down progression of disease. The proteinuria was reduced only in the iPS group. Both treatments reduced glomerulosclerosis, iPSs decreased macrophage infiltration, and TGF-ß was reduced in kidneys from the BMSC group. Both types of treatments increased VEGF gene expression, TGF-ß was upregulated only in the iPS group, and IL-10 had low expression in both groups. The SRY gene was found in 5/8 rats treated with iPSs. These 5 animals presented tumors with histology and cells highly staining positive for PCNA and Wilms' tumor protein antibody characteristics of Wilms' tumor. These results suggest that iPSs may be efficient to retard progression of CKD but carry the risk of Wilms' tumor development.

Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

Beneficial effects of prolactin and laminin on human pancreatic islet-cell cultures.

The problem of pancreas donor shortage could be addressed through in vitro islet-cell proliferation prior to transplantation into diabetic patients. Therefore, we set out to evaluate the effects of prolactin (rhPRL) and laminin on primary cultures of human pancreatic islets. Our results showed that rhPRL induced an increase in islet-cell number and in cumulative insulin secretion (p<0.01). However, glucose-induced insulin secretion was enhanced only in the presence of both laminin and rhPRL. In addition, we describe, for the first time in human islets, the PRL-induced activation of JAK2, and signal transducer and activator of transcription (STAT) 1, 3 and 5. Our results demonstrate a significant beneficial effect of rhPRL and laminin on human islets and support widely held notion that the closer physiological stimuli and environment of beta cells are mimicked, the better are the results in cell proliferation and secretory function, both essential for successful islet transplantation.

Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures.

Strategies to differentiate progenitor cells into beta cells in vitro have been considered as an alternative to increase beta cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell's fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional beta cells.