Confusion on Cell Fusion.

See rebuttal on page 307. See counterpoint on page 299.

Cell fusion is a key biologic process that underlies multicellular life. It is essential for fertilization when 2 gametes fuse; for placenta formation; and for organ development, including bone and muscle. Furthermore, cell fusion plays a role in immune responses when macrophages fuse to form multinucleated giant cells.

In recent years, researchers have set out to investigate the role of cell fusion in repair of damaged and diseased tissue. In particular, circulating bone marrow–derived cells (BMDCs) have received attention. One suggested mechanism of repair is that BMDCs fuse with stem cells or progenitors from the injured tissue to restore their function and maintain tissue integrity. This could involve the replacement of damaged organelles, the restoration of genetic material, and might assist in promoting dedifferentiation of more committed progenitors by nuclear reprogramming.

Indeed, experimental evidence for the occurrence of cell fusion between BMDCs and tissue resident cells in a wide variety of organs exists. These experiments usually involve the transplantation of bone marrow in mice. For example, BMDCs were shown to fuse with hepatocytes, with neurons, cardiac muscle, and intestinal epithelium. The role of cell fusion in intestinal epithelium has been particularly well-studied, likely given the tremendous regenerative potential and renewal observed in this tissue (Figure 1). However, possibly in contrast to other tissues that might rely on cell fusion for repair because of their relative quiescence (eg, Purkinje neurons or cardiomyocytes), we propose that the well-documented and extensive plasticity of the intestinal epithelial layer obliterates the need for cell fusion in this organ. Furthermore, although other tissues reported to depend on cell fusion for regeneration show evidence of widespread polyploidy (eg, liver), or fusion is an integral part of physiology already (eg, muscle), cells constituting these organs might indeed be susceptible to undergo fusion events with BMDCs. We think that current evidence suggests the intestine is not in this category.

Figure 1

Fusion or confusion in intestinal epithelium. Schematic representation of hypothetical cell fusion of intestinal cells with BMDCs and the confirmed mechanisms of repair because of intestinal plasticity. Intestinal epithelium is a tightly regulated and self-sustained tissue in homeostasis and disease. ISCs play a key role in maintaining the integrity of the intestinal layer. Differentiated cells, progenitors, and Paneth cells contribute to the regeneration of the crypt following damage. Cell fusion is therefore dispensible for the regeneration of the crypt.

Intestinal stem cells (ISCs), residing at the crypt bottoms, are key players in epithelial regeneration through their potential to continuously divide and replenish the stem cell pool and migrate out of the niche to differentiate., In 1 key intestinal cell fusion study by Rizvi et al, transplantation of GFP-expressing BMDCs from mice into R26R mice was performed. The model was tested in homeostasis and following irradiation. Microscopy analysis revealed a subset of cells in the intestinal crypt that expressed both GFP, as donor cell marker, and β-galactosidase, the host epithelium marker. It was concluded that this reveals the existence of cell fusion between BMDCs and intestinal epithelial cells, which seems to be boosted by injury and inflammation., However, the interpretation of these data remains debated because the model depends on continuous presence of genetic elements, both host and donor-derived in single cells, and could lead to false-positive results. These could both result from technical artefacts, and from the occurrence of other biologic phenomena, including horizontal gene transfer from apoptotic or circulating donor cells, and transdifferentiation of BDMCs to other cell lineages.8, 9, 10

To account for some of these shortcomings, other strategies are required that allow the detection of fusion through the exchange of genetic material. For example, we have aimed to resolve these issues by using Cre-inducible GFP, and Cre-inducible Diphteria Toxin Receptor mice (iGFP-iDTR), and transplanting them with Cre-expressing BMDCs. In the event of cell fusion, recombination would take place on the iDTR and iGFP loci marking those cells, and their offspring with GFP and DTR expression. Injection of diphtheria toxin results in the ablation of the fusion-derived GFP+/DTR+ cells. This system is highly sensitive to measure fusion events because only 1 of the reporter gene locus needs to be retained, whereas the Cre locus could be lost following the recombination event at the marker locus (iDTR or iGFP). The system was used to investigate the presence and functional role of cell fusion in the liver and the intestinal epithelium. We established the functionality of the model because hepatocytes showed a low but consistent fusion event frequency of 1:4000 cells. However, in our hands no convincing recombination took place in the intestinal epithelium. Furthermore, we adopted another model using iDTR mice transplanted with GFP+ bone marrow. In this experiment GFP+ cells could only be detected in the stromal compartment, demonstrating that also transdifferentiation of BMDCs into intestinal epithelium is indeed very rare, even after full-body irradiation with 8 Gy that is required to facilitate bone marrow transplantation. Induction of colitis in these mice by means of dextran sodium sulfate failed to markedly enhance the occurrence of cell fusion. Finally, ablation of fusion-derived DTR expressing cells and their offspring by treating colitis mice with diphtheria toxin did not result in impaired regeneration of the epithelial layer or in increased weight loss reflecting colitis severity. Combined, these data show that cell fusion between BMDCs and intestinal epithelial cells is very rare at best, and is dispensable for intestinal homeostasis and regeneration.

Instead, we advocate that the extensive epithelial cell plasticity observed in the intestine ensures that the tissue integrity is maintained, also in cases of extreme disruption. As previously mentioned, ISCs play an important role in tissue restoration., However, ISCs are not sole actors in the repair mechanism. Damage to the ISCs was shown to activate an adaptive regenerative response. Quiescent stem cells and progenitor cells from the secretory and absorptive lineages were all reported to contribute to replenish the ISCs pool (Figure 1).11, 12, 13, 14 For example, Buczacki et al investigated the role of quiescent label-retaining cells in regeneration. It was established that this population of committed Paneth cell precursors is able to dedifferentiate to reconstitute the ISC pool following doxorubicin-mediated damage. Similarly, enterocyte precursors and even Paneth cells can dedifferentiate following injury to generate ISCs., These studies highlight that various pools of epithelial cells with different degrees of differentiation can drive repair in the intestine. In an elegant recent study performed by the laboratory of Shivdasani, the Wnt target gene Ascl2 was demonstrated to be a principal factor for damage response. Deletion of Ascl2 hampered crypt regeneration following damage induction. Moreover, de novo expression of Ascl2 in more differentiated cells within the colonic crypt was used to detect and trace dedifferentiation and migration back to the ISCs niche. In this experimental system, dedifferentiation accounted for approximately 99% of restored ISCs, thus showing epithelial plasticity to be sufficient for repair. By extension, we conclude from these findings that cell fusion is dispensable. We think this notion is also corroborated by murine intestinal organoid cultures that display ongoing self-renewal potential for as long as 1.5 years, without any dependence on cell fusion.

In summary, although cell fusion might be a necessary process in other tissues, the gut is exquisitely well-balanced in both health and disease, and self-sustaining when it comes to tissue regeneration. ISCs and plasticity of progenitors and differentiated cells in combination with niche signals, harmoniously maintain the integrity of the intestinal layer (Figure 1). We think that the data to support this model of intestinal homeostasis and repair are extensive, and sufficient to explain experimental observations. As a result, it would require exceptionally strong evidence to claim that cell fusion is an integral part of gut biology, which we judge is lacking at this moment.