Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion. Mus musculus

Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion. Overall design: Examination was performed using single-cell RNA-seq of five fusion products (BiFC_D1_F1-5, 24 hours) identified using BiFC, twenty-three fusion products (DC_D1_F1-16, 24 hours; DC_D3_F1-7, 72 hours) identified using dual expression of GFP and mCherry, the parental controls, and the population controls (mMSC_PC and HL1cm_PC). Parental controls included 15 cells of each parental type isolated prior to co-culture (mMSC_1-15 and HL1cm_1-15) and 5 cells of each parental cell type isolated 24 hours after co-culture (mMSC_D1_1-5 and HL1cm_D1_1-5). In addition, a population containing a mixture of both parental cells and fusion products obtained 24 hours after co-culture was included (Mix_D1).

移植后，供体间充质干细胞与受体实质细胞可在脑、肝、肠道及心脏中发生融合。由此产生的杂交细胞的治疗获益与潜在危害目前尚不明确。本研究旨在全面解析间充质干细胞-心肌细胞杂交细胞的表型分化特征及其伴随的时间进程。
本研究采用单细胞RNA测序（single-cell RNA-seq）技术，发现杂交细胞普遍呈现核糖体组分基因表达上调、细胞周期相关基因表达下调的特征，提示其为适应融合状态，蛋白质合成增加而增殖能力降低。但就绝大多数其他基因类别而言，各杂交细胞个体间均存在显著差异。
事实上，尽管部分杂交细胞的转录组可与单一融合亲本相似，但约有三分之一的杂交细胞可在单日之内获得独特的表达谱。部分杂交细胞发生重编程，表达亲本细胞中潜在存在的多能性及心脏前体细胞基因，且这些基因与发育及形态发生相关基因簇相关联。另有部分杂交细胞表达与肿瘤相关基因集相关的基因；在独立实验重复中获得的两株杂交细胞可与乳腺癌细胞聚类，其表达关键癌基因但缺失抑癌基因。
此类快速转录组分化现象，在向受损组织、病毒感染组织或其他可能促进细胞融合的微环境中进行细胞移植时，值得重点关注。

整体实验设计：本研究通过单细胞RNA测序完成相关检测，样本包括：
1. 利用双荧光互补技术（bimolecular fluorescence complementation, BiFC）鉴定得到的5株融合产物（BiFC_D1_F1-5，培养24小时）；
2. 利用绿色荧光蛋白（green fluorescent protein, GFP）与红色荧光蛋白mCherry双表达标记鉴定得到的23株融合产物，其中培养24小时组为DC_D1_F1-16，培养72小时组为DC_D3_F1-7；
3. 亲本对照：共培养前分离的两种亲本细胞各15株（mMSC_1-15与HL1cm_1-15），以及共培养24小时后分离的两种亲本细胞各5株（mMSC_D1_1-5与HL1cm_D1_1-5）；
4. 群体对照：mMSC_PC与HL1cm_PC；
5. 混合样本：共培养24小时后收集的亲本细胞与杂交细胞混合群体（Mix_D1）。