Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

Reyes, Álvaro Plaza; Petrus‐Reurer, Sandra; Sánchez, Sara Padrell; Kumar, Pankaj; Douagi, Iyadh; Bartuma, Hammurabi; Aronsson, Monica; Westman, Sofie; Lardner, Emma; André, Helder; Falk, Anna; Nandrot, Emeline F.; Kvanta, Anders; Lanner, Fredrik

doi:10.1038/s41467-020-15326-5

Cited by 33 publications

(21 citation statements)

References 40 publications

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“…We further acknowledge that the RPE cell models we employed may contain small subsets cells in a variably differentiated state that have a transcriptional landscape deviating from that of the fully differentiated population. To characterize this further experiments utilizing multiplex flow cytometry to directly link markers of RPE differentiation (e.g., Plaza Reyes et al, 2020) to synthetic promoter mediated reporter gene expression would be possible.…”

Section: Discussionmentioning

confidence: 99%

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

Johari

Mercer

Liu

et al. 2021

Biotech & Bioengineering

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Age‐related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE‐specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre‐defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8–19 bp) specifically associated with transcriptionally active RPE genes. Both RPE‐specific TFREs and those derived from the generically active cytomegalovirus‐immediate early (CMV‐IE) promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)‐derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323–602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV‐IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8‐fold that of the RPE‐specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV‐IE promoter when viral elements were utilized in combination with endogenous RPE‐specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell‐type specificity, cell context‐specific control of recombinant gene transcriptional activity may be achievable.

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Section: Discussionmentioning

confidence: 99%

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

Johari

Mercer

Liu

et al. 2021

Biotech & Bioengineering

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“…Another possibility is that our iPSC-RPE were insufficiently matured, which delayed the concomitant cellular events. In that regard, efforts for quantitative quality control to assess maturity instead of image-based analyses are emerging ( Plaza Reyes et al, 2020 ). Parallel studies with both genetic backgrounds are warranted in order to delineate the specific roles of each PRPF gene.…”

Section: Discussionmentioning

confidence: 99%

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells

et al. 2021

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Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.

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“…Erdogan et al reported that AT-MSCs in New Zealand rabbits did not express CD73 and CD90 [ 58 ], while Chen et al detected the expression of CD90 [ 59 ]. Some markers appeared already at the optic vesicle stage but did not remain highly expressed in the later differentiation stage [ 74 ].…”

Section: Mscs and Mscs-exosomes: Cell Selection And Preparationmentioning

confidence: 99%

Mesenchymal stromal cell-based therapy for cartilage regeneration in knee osteoarthritis

Xiang

Zhu

et al. 2022

Stem Cell Res Ther

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Osteoarthritis, as a degenerative disease, is a common problem and results in high socioeconomic costs and rates of disability. The most commonly affected joint is the knee and characterized by progressive destruction of articular cartilage, loss of extracellular matrix, and progressive inflammation. Mesenchymal stromal cell (MSC)-based therapy has been explored as a new regenerative treatment for knee osteoarthritis in recent years. However, the detailed functions of MSC-based therapy and related mechanism, especially of cartilage regeneration, have not been explained. Hence, this review summarized how to choose, authenticate, and culture different origins of MSCs and derived exosomes. Moreover, clinical application and the latest mechanistical findings of MSC-based therapy in cartilage regeneration were also demonstrated.

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Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

Cited by 33 publications

References 40 publications

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells

Mesenchymal stromal cell-based therapy for cartilage regeneration in knee osteoarthritis

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