Chen, F. K. | bioRxiv Science

bioRxiv2024

Accelerated Limbal Epithelial Differentiation of Human Induced Pluripotent Stem Cells Using a Defined Keratinocyte Serum-Free Medium

PurposeTreatment of bilateral limbal stem cell deficiency (LSCD) is challenging due to the limited autologous stem cell sources. This study aimed to differentiate human induced pluripotent stem cells (hiPSCs) into limbal epithelial stem cells (LESCs) using a defined keratinocyte serum-free medium (DKSFM). MethodsA fully characterized hiPSC line was committed to ectodermal differentiation using Essential 6 (E6) medium supplemented with 10 {micro}M Y-27632 (Day 1), 10 {micro}M SB-505124 plus 50 ng/ml bFGF (Day 2) and 25 ng/ml BMP-4 (Days 3 and 4). Differentiation was continued in DKSFM for an additional 21 days. Quantitative PCR (qPCR) and/or immunocytochemistry (ICC) for pluripotency, proliferation, LESC, and corneal epithelial markers were performed on samples collected at days 5, 10, 15, and 25 (D5 to D25) and compared with undifferentiated hiPSCs (UD). ResultsqPCR revealed a significant decrease in the expression of OCT4 and NANOG and a significant increase in ABCG2 and TP63 following ectodermal induction (i.e., D5), compared with UD (P < 0.05). The expression levels of Ki67, ABCG2, TP63, and CK14 were significantly higher at D10, compared with D5 and D25 (P < 0.05). The ratio of p63-positive cells was 71% and 56% in D10 and D15 cells, respectively (P < 0.05). DiscussionOur method resulted in a limited but rapid differentiation of hiPSCs into LESC-like cells. The LESC-like cells appeared as early as 5 days following ectodermal induction and their population peaked after 10 days. Upon further optimization and validation, DKSFM can be used for rapid limbal epithelial differentiation of hiPSCs.

bioengineering↗

bioRxiv2024

Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduce ABCA4 splice variants in Stargardt disease

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

molecular biology↗