Myeloid-like tumor hybrid cells in bone marrow promote progression of prostate cancer bone metastasis.

BACKGROUND: Bone metastasis is the leading cause of death in patients with prostate cancer (PCa) and currently has no effective treatment. Disseminated tumor cells in bone marrow often obtain new characteristics to cause therapy resistance and tumor recurrence. Thus, understanding the status of disseminated prostate cancer cells in bone marrow is crucial for developing a new treatment. METHODS: We analyzed the transcriptome of disseminated tumor cells from a single cell RNA-sequencing data of PCa bone metastases. We built a bone metastasis model through caudal artery injection of tumor cells, and sorted the tumor hybrid cells by flow cytometry. We performed multi-omics analysis, including transcriptomic, proteomic and phosphoproteomic analysis, to compare the difference between the tumor hybrid cells and parental cells. In vivo experiments were performed to analyze the tumor growth rate, metastatic and tumorigenic potential, drug and radiation sensitivity in hybrid cells. Single cell RNA-sequencing and CyTOF were performed to analyze the impact of hybrid cells on tumor microenvironment. RESULTS: Here, we identified a unique cluster of cancer cells in PCa bone metastases, which expressed myeloid cell markers and showed a significant change in pathways related to immune regulation and tumor progression. We found that cell fusion between disseminated tumor cells and bone marrow cells can be source of these myeloid-like tumor cells. Multi-omics showed the pathways related to cell adhesion and proliferation, such as focal adhesion, tight junction, DNA replication, and cell cycle, were most significantly changed in these hybrid cells. In vivo experiment showed hybrid cells had a significantly increased proliferative rate, and metastatic potential. Single cell RNA-sequencing and CyTOF showed tumor-associated neutrophils/monocytes/macrophages were highly enriched in hybrid cells-induced tumor microenvironment with a higher immunosuppressive capacity. Otherwise, the hybrid cells showed an enhanced EMT phenotype with higher tumorigenicity, and were resistant to docetaxel and ferroptosis, but sensitive to radiotherapy. CONCLUSION: Taken together, our data demonstrate that spontaneous cell fusion in bone marrow can generate myeloid-like tumor hybrid cells that promote the progression of bone metastasis, and these unique population of disseminated tumor cells can provide a potential therapeutic target for PCa bone metastasis.

A genome on shaky ground: exploring the impact of mitochondrial DNA integrity on Parkinson's disease by highlighting the use of cybrid models.

Mitochondria play important roles in the regulation of key cellular processes, including energy metabolism, oxidative stress response, and signaling towards cell death or survival, and are distinguished by carrying their own genome (mtDNA). Mitochondrial dysfunction has emerged as a prominent cellular mechanism involved in neurodegeneration, including Parkinson's disease (PD), a neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons and the occurrence of proteinaceous Lewy body inclusions. The contribution of mtDNA variants to PD pathogenesis has long been debated and is still not clearly answered. Cytoplasmic hybrid (cybrid) cell models provided evidence for a contribution of mtDNA variants to the PD phenotype. However, conclusive evidence of mtDNA mutations as genetic cause of PD is still lacking. Several models have shown a role of somatic, rather than inherited mtDNA variants in the impairment of mitochondrial function and neurodegeneration. Accordingly, several nuclear genes driving inherited forms of PD are linked to mtDNA quality control mechanisms, and idiopathic as well as familial PD tissues present increased mtDNA damage. In this review, we highlight the use of cybrids in this PD research field and summarize various aspects of how and to what extent mtDNA variants may contribute to the etiology of PD.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model.

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid 'OLL8' sweet orange or 'Page' tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the 'OLL8' + FL fusion, while three putative cybrids were regenerated from the 'Page' + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Cell fusion enhances energy metabolism of mesenchymal tumor hybrid cells to sustain their proliferation and invasion.

BACKGROUND: Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. RESULTS: Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-ß-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. CONCLUSION: Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy.

Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mdx mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MBN)] and normal human bone marrow-derived MSC (MSCN) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MBN/MSCN DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MBN/MSCN DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MBN and MSCN (n = 9, 0.25 × 106/each) or MBN/MSCN DEC (n = 9, 0.5 × 106). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MBN/MSCN DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MBN/MSCN DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.

Differential effects of risuteganib and bevacizumab on AMD cybrid cells.

PURPOSE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). METHODS: Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 µl (25,000 µg/ml) of Bmab or 1.0 mg/50 µl (20,000 µg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1ß, IFN-ß1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. RESULTS: The untreated AMD cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal cybrids. The Bmab-treated AMD cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD cybrids. The Bmab-treated cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated cybrids had higher expression levels of inflammatory (IL-6, IL1-ß), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. CONCLUSIONS: These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.

Cell Fusion of Mesenchymal Stem/Stromal Cells and Breast Cancer Cells Leads to the Formation of Hybrid Cells Exhibiting Diverse and Individual (Stem Cell) Characteristics.

Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological (e.g., wound healing) and pathophysiological (cancer and viral infection) processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry (CD24, CD44, CD104) and Western blot analysis (of epithelial to mesenchymal transition markers such as SNAIL, SLUG and Twist) were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Altered Tumor Plasticity after Different Cancer Cell Fusions with MSC.

While cell fusion demonstrates an important pathway during tissue development and regeneration of distinct organs, this process can also contribute to pathophysiological phenotypes during tumor progression. Hybrid cell formation after heterofusion between cancer cells and various other cell types within the tumor microenvironment is observed in vitro and in vivo. In particular, mesenchymal stroma/stem-like cells (MSC) perform diverse levels of communication with cancer cells by exhibiting anti- and pro-tumorigenic effects. During these cellular interactions, MSC can eventually fuse with cancer cells. Thereby, the newly generated disparate hybrid populations display aneuploidy associated with chromosomal instability. Based upon a subsequent post-hybrid selection process (PHSP), fused cancer cells can undergo apoptosis/necroptosis, senescence, dormancy, or a proliferative state by acquisition of new properties. Consequently, PHSP-surviving hybrid cancer cells demonstrate altered functionalities within the tumor tissue. This is accompanied by changes in therapeutic responsiveness and a different metastatic behavior. Accordingly, enhanced tumor plasticity interferes with successful therapeutic interventions and aggravates patient prognoses. The present review article focusses on fusion of MSC with different human cancer cells, in particular breast cancer populations and resulting characteristics of various cancer hybrid cells. Moreover, some mechanisms of cancer cell fusion are discussed together with multiple PHSP pathways.

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes.

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

Sequenced Somatic Cell Reprogramming and Differentiation Inside Nested Hydrogel Droplets.

The efficient genesis of pluripotent cells or therapeutic cells for regenerative medicine involves several external manipulations and conditioning protocols, which drives down clinical applicability. Automated programming of the genesis by microscale physical forces and chronological biochemistry can increase clinical success. The design and fabrication of nested polysaccharide droplets (millimeter-sized) with cell sustaining properties of natural tissues and intrinsic properties for time and space evolution of cell transformation signals between somatic cells, pluripotent cells and differentiated therapeutic cells in a swift and efficient manner without the need for laborious external manipulation are reported. Cells transform between phenotypic states by having single and double nested droplets constituted with extracellular matrix proteins and reprogramming, and differentiation factors infused chronologically across the droplet space. The cell transformation into germ layer cells and bone cells is successfully tested in vitro and in vivo and promotes the formation of new bone tissues. Thus, nested droplets with BMP-2 loaded guests synthesize mineralized bone tissue plates along the length of a cranial non-union bone defect at 4 weeks. The advantages of sequenced somatic cell reprogramming and differentiation inside an individual hydrogel module without external manipulation, promoted by formulating tissue mimetic physical, mechanical, and chemical microenvironments are shown.

Comparison of hybrid clones derived from human breast epithelial cells and three different cancer cell lines regarding in vitro cancer stem/ initiating cell properties.

BACKGROUND: Several physiological (fertilization, placentation, wound healing) and pathophysiological processes (infection with enveloped viruses, cancer) depend on cell fusion. In cancer it was postulated that the fusion of cancer cells with normal cells such as macrophages or stem cells may not only give rise to hybrid cells exhibiting novel properties, such as an increased metastatic capacity and drug resistance, but possibly also cancer stem/ initiating cell properties. Hence, hybrid clone cells (M13HS, M13MDA435 and M13MDA231) that were derived from spontaneous fusion events of human M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg, MDA-MB-435-Hyg and MDA-MB-231-Hyg cancer cells were investigated regarding potential in vitro cancer stem/ initiating cell properties. METHODS: CD44/CD24 expression pattern and ALDH1 activity of parental cells and hybrid clones was determined by flow cytometry. A colony formation and mammosphere formation assay was applied to determine the cells' capability to form colonies and mammospheres. Sox9, Slug and Snail expression levels were determined by Western blot analysis. RESULTS: Flow cytometry revealed that all hybrid clone cells were CD44+/CD24-/low, but differed markedly among each other regarding ALDH1 activity. Likewise, each hybrid clone possessed a unique colony formation and mammosphere capacity as well as unique Snail, Slug and Sox9 expression patterns. Nonetheless, comparison of hybrid clones revealed that M13HS hybrids exhibited more in vitro cancer stem/ initiating cell properties than M13MDA231 and M13MDA435 hybrids, such as more ALDH1 positive cells or an increased capacity to form colonies and mammospheres. CONCLUSION: The fate whether cancer stem/ initiating cells may originate from cell fusion events likely depends on the specific characteristics of the parental cells.

Pioglitazone and Deoxyribonucleoside Combination Treatment Increases Mitochondrial Respiratory Capacity in m.3243A>G MELAS Cybrid Cells.

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.

Cybrid technology.

The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids.

Mitochondrial DNA Haplotypes Influence Energy Metabolism across Chicken Transmitochondrial Cybrids.

The association between mitochondrial DNA haplotype and productive performances has been widely reported in chicken breeds. However, there has not been physiological evidence of this seen previously. In this study, chicken transmitochondrial cells were generated using the nucleus of the DF-1 cell line and mitochondria of primary cell lines derived from two native chicken breeds, Tibetan chicken and Shouguang chicken. Generally, Tibetan chicken primary cells showed a stronger metabolic capacity than Shouguang chicken primary cells. However, the Tibetan chicken cybrids had a dramatic drop in relative mtDNA copies and oxygen consumption. Higher rates of oxygen consumption (OCR) and expression levels of mitochondrial biogenesis and fusion genes were observed in Shouguang chicken cybrids, potentially reflecting that the mitochondrial DNA haplotype of Shouguang chicken had better coordination with the DF-1 nucleus than others. Meanwhile, mitonuclear incompatibility occurred in Tibetan chicken cybrids. The results demonstrate functional differences among mitochondrial DNA haplotypes and may shed light on the interaction between the mitochondria and nucleus in Gallus gallus domesticus.

Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes.

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.

Fusion-mediated chromosomal instability promotes aneuploidy patterns that resemble human tumors.

Oncogenesis is considered to result from chromosomal instability, in addition to oncogene and tumor-suppressor alterations. Intermediate to aneuploidy and chromosomal instability, genome doubling is a frequent event in tumor development but the mechanisms driving tetraploidization and its impact remain unexplored. Cell fusion, one of the pathways to tetraploidy, is a physiological process involved in mesenchymal cell differentiation. Besides simple genome doubling, cell fusion results in the merging of two different genomes that can be destabilized upon proliferation. By testing whether cell fusion is involved in mesenchymal oncogenesis, we provide evidence that it induces genomic instability and mediates tumor initiation. After a latency period, the tumor emerges with the cells most suited for its development. Furthermore, hybrid tumor genomes were stabilized after this selection process and were very close to those of human pleomorphic mesenchymal tumors. Thus genome restructuring triggered by cell fusion may account for the chromosomal instability involved in oncogenesis.

Generation and maintenance of acentric stable double minutes from chromosome arms in inter-species hybrid cells.

BACKGROUND: Extrachromosomal acentric double minutes (DMs) contribute to human malignancy by carrying amplified oncogenes. Recent cancer genomics revealed that the pulverization of defined chromosome arms (chromothripsis) may generate DMs, however, nobody had actually generated DMs from chromosome arm in culture. Human chromosomes are lost in human-rodent hybrid cells. RESULTS: We found that human acentric DMs with amplified c-myc were stable in human-rodent hybrid cells, although the degree of stability depended on the specific rodent cell type. Based on this finding, stable human-rodent hybrids were efficiently generated by tagging human DMs with a plasmid with drug-resistance gene. After cell fusion, human chromosomes were specifically pulverised and lost. Consistent with chromothripsis, pulverization of human chromosome arms was accompanied by the incorporation into micronuclei. Such micronucleus showed different replication timing from the main nucleus. Surprisingly, we found that the hybrid cells retained not only the original DMs, but also new DMs without plasmid-tag and c-myc, but with human Alu. These DMs were devoid of telomeres and centromeres, and were stable in culture for more than 3 months. Microarray analysis showed that the new DMs were generated from several human chromosomal regions containing genes advantageous for cellular growth. Such regions were completely different from the original DMs. CONCLUSIONS: The inter-species hybrid mimics the chromothripsis in culture. This is the first report that experimentally demonstrates the generation of multiple stable acentric DMs from the chromosome arm.

In vitro differentiation of mouse T cell-derived hybrid cells obtained through cell fusion with embryonic stem cells.

Nuclear reprogramming is an innovative advance in cell biology. An important research initiative in this field is cell fusion-mediated nuclear reprogramming, wherein the nuclei of somatic cells, such as thymocytes, are initialized through cell fusion with embryonic stem cells (ESCs). However, hybrid cells obtained through cell fusion between ESCs and thymocytes failed to contribute to the embryo proper when injected into blastocysts, which suggested that there are fundamental defects in such hybrid cells. Here, we performed side-by-side comparative analyses of the in vitro growth and differentiation capacities of ESCs and ESC-T hybrid cells. We found that the hybrid cells were larger and proliferated more slowly than the ESCs in 2i/LIF medium. Upon in vitro induction of differentiation, hybrid cells gave rise to cells of the three germ layers. Under culture conditions for hematopoietic differentiation, hybrid cells successively differentiated into lateral mesodermal cells, hemogenic endothelial cells, and various types of hematopoietic cells, including erythroid, myeloid, and lymphoid cells, although T cell maturation in the CD4/CD8 double-negative fraction was delayed. These results verified the multi-lineage differentiation capacity of ESC-T hybrid cells. The minimal contribution of hybrid cells to chimeric embryos may be due to their slow growth.