Edel, M. J. | Search bioRxiv

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↗

bioRxiv2022

Immunosuppressive tocilizumab prevents astrocyte induced neurotoxicity in hiPSC-LRRK2 Parkinson's disease by targeting receptor interleukin-6

Parkinsons disease (PD) is associated with premature death of dopamine-producing neurons in the brain. Previous studies have shown that astrocytes of PD patients may contribute to neuronal degeneration by mechanisms involving both direct cell-to-cell contact and transfer of soluble molecules. Since it has been proposed that PD patients exhibit an overall pro-inflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing PD patients and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared to controls. In particular, the pro-inflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes, and to induce toxicity in dopamine neurons. Mechanistically, neuronal cell death was mediated by IL-6 signaling via IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Importantly, astrocyte-induced cell death in PD disease midbrain neurons could be prevented by blocking IL6R-mediated signaling using clinically available antibodies. Moreover, examination of postmortem tissue brain of early-stage PD patients uncovered increased numbers of dopamine neurons overexpressing IL-6R and of reactive astrocytes overexpressing IL-6, compared to healthy brains. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD, and open the way for new therapies based on IL-6 immunomodulation for preventing PD pathogenesis.

neuroscience↗