PREVENTION OF NUCLEAR MIXING OF FUSED CELLS INTERCONNECTED THROUGH A MICROSLIT USING ROCK INHIBITOR Y-27632 FOR DIRECT CYTOPLASMIC TRANSFER
BETWEEN LIVE SINGLE CELLS K.-I. Wada*, K. Hosokawa, Y. Ito and M. Maeda
RIKEN, Japan
ABSTRACT We previously reported a cell fusion method using a microfluidic device, in which paired single cells were fused through a microslit [1]. Although our approach realized direct cytoplasmic transfer between live single cells and thus is a promising method to induce cellular reprogramming or transdifferentiation, prevention of nuclear mixing with the microslit was unsatisfactory (~15%). We herein report that the nuclear mixing was almost perfectly prevented (~95%) by addition of ROCK inhibitor Y-27632 in NIH3T3 fibroblast. Furthermore, Y-27632 raised the probability of cell fusion through the microslit. As a result, we obtained 16.5-fold increase in the number of nuclear-separated fused cell pairs. KEYWORDS: Microslit, Cell fusion, Y-27632, Cytoplasmic transfer, Mitochondria
INTRODUCTION Cell fusion is a promising method to produce cellular resources for regenerative medicine and cell therapy because of the potential to induce phenotypic changes such as transdifferentiation and reprogramming due at least partly to cytoplasmic transfer between fused cells [2, 3]. However, common cell fusion methods produce nuclear mixing that results in abnormal karyotypes. If cell fusion can be induced without nuclear mixing, this approach will be a powerful tool for transferring of cytoplasm to induce transdifferentiation and reprogramming.
Recently, we succeeded in inducing cell fusion through a microslit between adhered/spread cells by using a newly developed microfluidic device [1]. Although our cell fusion method realized direct cytoplasmic transfer without nuclear mixing, it was still impractical because of the low yield of cell fusion induction through a microslit without nuclear mixing. With our method, less than 10% of cell pairs underwent cell fusion through a microslit. Furthermore, observations of the fused cells showed that their nuclei migrated into the fusion partner passing through the microslit and resulted in nuclear mixing. As a consequence, only 1.3% of cell pairs formed a strictured cytoplasmic connection without nuclear mixing. Therefore, it was necessary to improve the cell fusion properties in order to make our cell fusion system practical.
Here we describe that supplementation with Y-27632, a specific ROCK (Rho-associated coiled coil-forming protein kinase) inhibitor, which induces stress fiber disruption, led to a 16.5-fold improvement in the formation of fused cell pairs without nuclear mixing. We also demonstrate that the Y-27632 supplementation protocol could achieve mitochondrial transfer to the fusion partner. Therefore, Y-27632 supplementation is a promising protocol that can be used to make our cell fusion system practical.
THEORY After making cells pair in a microfabricated device, cell fusion is induced through a small aperture termed “microslit” to make a strictured cytoplasmic connection. We previously found that this approach prevents nuclear migration to the fusion partner between adhered cells, but the nucleus in a round cell migrates into the fusion partner passing through the microslit, resulting in nuclear mixing (Fig. 1). Since cytoskeletons play a role in nuclear positioning, we hypothesized that modulation of cytoskeletal component(s) can prevent nuclear migration. To test this hypothesis, we carried out experiments involving the pharmacological disruption of F-actin by specific inhibitors.
Figure 1: Cell fusion system for cytoplasmic transfer. (A) Photograph of the cell fusion system. Microfluidic channel, which has 105 cell paring structures (CPSs) is constructed on a 35-mm cell culture dish. Microslit (arrow) is fabricated in the separation wall of CPS at the position of paired cell-trap pocket. (B) Schematic illustration of cytoplasmic transfer by cell fusion through a microslit. In our previous trial, nuclear mixing occurred in round cells [1].
EXPERIMENTAL A poly(dimethylsiloxane) (PDMS) chip, which had 105 cell pairing structures (CPSs) in the microchannel with a width of 500 μm and a depth of 28 μm, was fabricated using a photolithography-based method. The PDMS chip was then bonded on the bottom of a 35-mm cell culture dish to prepare a microfluidic device. The NIH3T3 fibroblast-derived cell lines Ng3T3, CrNr3T3, and Mito3T3 were established by stable transfection of H2B-EGFP, H2B-EGFP/H2B-mCherry, and COX8a-EGFP expression vector(s), respectively. To induce cell fusion through a microslit, 100 μL fusion medium containing Sendai virus envelope (HVJ-E) (Ishihara Sangyo Kaisha Ltd.) was applied to the inlet reservoir after cells were trapped in CPSs. For disruption of cytoskeletal components, Cytochalasin D (CytoD), Latrunculin A (LatA), or Y-27632 was added to culture medium. The fluorescent signals of EGFP and mCherry were observed directly using reverse fluorescent microscopy.
RESULTS AND DISCUSSION Supplementation with 2 μM CytoD or 1 μM LatA resulted in cellular rounding up. As a result, cell fusion through the microslit was formed between round cells. However, interestingly, the nuclei in these cells did not move into the fusion partner (data not shown), even though our previous study showed that the nuclei in round cells moved to the fusion partner through the microslit [1]. Similar to CytoD and LatA, expectedly, Y-27632 also showed inhibitory effect on nuclear mixing (Fig. 2A). In the most effective case (i.e., supplementation with 160 μM Y-27632), 94.9% of the fused cells showed nuclear separation. These results suggest that F-actin is involved with nuclear migration to the fusion partner passing through the microslit. Added to this, Y-27632 supplementation increased the frequency of cell fusion through the microslit by more than 2 fold (Fig. 2A “Fusion total”). Due to these two distinct effects, the Y-27632 supplementation protocol promoted fused cell pairs without nuclear mixing with a 16.5-fold higher efficiency compared to Y-27632 absent protocol (Fig. 2A “Separated”).
Due to disruption of F-actin, supplementation of Y-27632 would induce dysfunction of the actomyosin-associated transporting system. Therefore, it was doubtful whether cytoplasmic transfer was realized under a condition of supplementation with Y-27632. To confirm the occurrence of functional cytoplasmic transfer, we performed live cell imaging on mitochondria as a model of insoluble cytoplasmic component, and revealed that mitochondria were successfully transferred into the fusion partner through the microslit even under a condition of 160 μM Y-27632 supplementation (Fig. 2B). During mitochondrial transferring, nuclear mixing did not occur. These data suggest that the Y-27632 supplementation protocol can achieve direct cytoplasmic transfer without nuclear mixing between live single cells in our fusion system.
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CONCLUSION We found unique properties of Y-27632, which make our cell fusion system practical for the realization of direct cytoplasmic transfer. Our findings will contribute significantly to the development of effective and productive cellular resources applicable to regenerative medicine and cell biology.
Figure 2: Effects of Y-27632 supplementation. (A) Prevention of nuclear mixing and enhancement of cell fusion through the microslit. Nuc: nucleus of Ng3T3 cell (green). mCh: nucleus and cytoplasmic area of CrNr3T3 cells (red). Ph: phase-contrast. *Significantly different from 0 μM (P < 0.05). (B) Mitochondria were transferred to the fusion partner under condition of 160 μM Y-27632 supplementation.
ACKNOWLEDGEMENT We thank the Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Bi-ology, RIKEN Kobe for providing expression vectors.
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