A Cell Electrofusion Chip for Somatic Cells Reprogramming

Wu, Wei; Qu, Ya; Hu, Ning; Zeng, Yuxiao; Yang, Jun; Xu, Haiwei; Yin, Zheng

doi:10.1371/journal.pone.0131966

Cited by 13 publications

(11 citation statements)

References 43 publications

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“…U6 snRNA was used as an endogenous control for miRNA detection. RT-qPCR analysis for BDNF and mGluR6 mRNA was performed according to previously described methods 13 . Briefly, total RNA was reverse transcribed using a PrimeScript® RT Reagent Kit (Takara Bio USA, Mountain View, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats

Hou

Weng

et al. 2017

Sci Rep

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Following retinal degeneration, retinal remodeling can cause neuronal microcircuits to undergo structural alterations, which particularly affect the dendrites of bipolar cells. However, the mechanisms and functional consequences of such changes remain unclear. Here, we used Royal College of Surgeon (RCS) rats as a model of retinal degeneration, to study structural changes in rod bipolar cells (RBCs) and the underlying mechanisms of these changes. We found that, with retinal degeneration, RBC dendrites extended into the outer nuclear layer (ONL) of the retina, and the ectopic dendrites formed synapses with the remaining photoreceptors. This ectopic neuritogenesis was associated with brain-derived neurotrophic factor (BDNF) – expression of which was negatively regulated by miR-125b-5p. Overexpression of miR-125b-5p in the retinae of RCS rats diminished RBC ectopic dendrites, and compromised the b-wave of the flash electroretinogram (ERG). In contrast, down-regulation of miR-125b-5p (or exogenous BDNF treatment) increased RBC ectopic dendrites, and improved b-wave. Furthermore, we showed that the regulation of ectopic neuritogenesis by BDNF occurred via the downstream modulation of the TrkB-CREB signaling pathway. Based on these findings, we conclude that ectopic dendrites are likely to be providing functional benefits and that, in RCS rats, miR-125b-5p regulates ectopic neuritogenesis by RBCs through modulation of the BDNF-TrkB-CREB pathway. This suggests that therapies that reduce miR-125b-5p expression could be beneficial in human retinal degenerative disease.

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Section: Methodsmentioning

confidence: 99%

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats

Hou

Weng

et al. 2017

Sci Rep

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“…Based on the number of aligned versus fused cell pairs, we determined the fusion efficiency to be more than 90%, proving the capability of our device for high-yield one-to-one electrofusion. Overall, our one-to-one fusion via micro-slit technique achieves one-to-one fusion with high efficiency compared to conventional bulk electrofusion where cells in the pearl chain are randomly fused 13 . In addition, since electric-field constriction at the micro-slits amplifies an effective voltage, fusion can be achieved at a low voltage of ∼10 V thus minimizing cell damage.…”

Section: Resultsmentioning

confidence: 93%

“…Although various studies have attempted fusion in a microfluidic system, 13,20,21 subsequent on-chip culture and imaging has proved to be challenging and only attempted by a few studies 12,22 . The challenges of continuous culture inside a PDMS device are well documented 23 .…”

Section: Discussionmentioning

confidence: 99%

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids

et al. 2016

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Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 μm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2–3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

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“…qPCR was carried out as previously described [39]. In brief, total RNA of each retina was extracted with 1 mL TRIzol Reagent (Sigma), 200 µL chloroform, 500 µL isopropanol, and 1 mL 75% ethyl alcohol.…”

Section: Methodsmentioning

confidence: 99%

Curcumin Delays Retinal Degeneration by Regulating Microglia Activation in the Retina of rd1 Mice

Wang

Yin

Gao

et al. 2017

Cell Physiol Biochem

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Background/Aims: Retinitis pigmentosa (RP) is characterized by degeneration of photoreceptors, and there are currently no effective treatments for this disease. However, curcumin has shown neuroprotectant efficacy in a RP rat and swine model, and thus, may have neuroprotective effects in this disease. Methods: Immunofluorescence staining, electroretinogram recordings, and behavioral tests were used to analyze the effects of curcumin and the underlying mechanism in retinal degeneration 1 (rd1) mice. Results: The number of apoptotic cells in the retina of rd1 mice at postnatal day 14 significantly decreased with curcumin treatment and visual function was improved. The activation of microglia and secretion of chemokines and matrix metalloproteinases in the retina were inhibited by curcumin. These effects were also observed in a co-culture of BV2 microglial cells and retina-derived 661W cells. Conclusions: Curcumin delayed retinal degeneration by suppressing microglia activation in the retina of rd1 mice. Thus, it may be an effective treatment for neurodegenerative disorders such as RP.

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A Cell Electrofusion Chip for Somatic Cells Reprogramming

Cited by 13 publications

References 43 publications

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids

Curcumin Delays Retinal Degeneration by Regulating Microglia Activation in the Retina of rd1 Mice

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