The potential of cell fusion for human therapy

Stem Cell Reviews Copyr ight �9 20fl6 Humana Press Inc. All r ights of any nature whatsoever are reserved. ISSN 1535-1084/06/2:341-350/$30.00 (Online) 1558-6804

The Potential of Cell Fusion for Human Therapy Stephen Sullivan and Kevin Eggan*

Stowers Medical Institute, Harvard Stem Cell Institute, and Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA

Abstract As donor organs and tissues for transplantation medicine are scarce, alternative methods for replacing damaged cells or restoring organ function are highly needed. Here, we consider the therapeutic potential of cell fusion. After highhghting the various contexts in which cells are known to fuse during mammalian development, we discuss the implica- tions of the observation that cell fusion can occur with restorative effects following tissue damage or cell transplantation. There are still, however, many challenges facing those who wish to implement cell fusion as a therapeutic tool. These include identifying the best cells to use for reparative fusion, determining the best route of introducing these cells into the desired tissue, discovering methods to increase the incidence of cell fusion, and ensuring the functionality of the resulting fusion products. If these difficulties can be overcome, cell fusion might have therapeutic potenbal as highlighted by several recent transplantation studies.

Index Entries: Differentiation; epigenetic; fusion; heterokaryon; hybrid; nuclear transfer; phenoty pic dominance; reprogrammmg; stem cell; synkaryon; tetra ploid therapy; trans- &fferentiation; transplant.

*Correspondence and reprint requests to:

Kevin Eggan, Stowers Me&cal Institute, Harvard Stem Cell Institute, and Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambndge, MA 02138, USA E-mail: [email protected]

H u m a n a Press

Introduction Cel l t heo ry , i n i t i a l l y a r t i c u l a t e d by

Schleiden and Schwann (1839) states that cells are dist inct , self-defined units from which all life is made (1,2). However , this theory also takes into account that certain cells fuse wi th one another (3), and such fusion is necessary for normal deve lopment of m u l t i c e l l u l a r o r g a n i s m s , i n c l u d i n g mammals (Fig. 1). Mammal ian cell fusion is essential for processes such as fertilization (Fig. 1A) and the development of a range of tissues (Fig. 1B-D; 4).

In th is r ev iew, we s u m m a r i z e the instances in which cell fusion is known to occur "spontaneously" in vitro as well as in vivo (during development , cellular differentiation, or following cell transplantation). We also consider ways in which damaged or genetically deficient somatic cells could be rescued by fusion with stem cells or their

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derivatives. The main obstacles facing the use of cell fusion for therapies are listed, and we speculate on how they might be overcome.

Molecular Control and Mechanistic Aspects of Cell Fusion

Although cell fusion was first observed by Schwann in vivo as early as 1847, it has general ly been assumed that this process plays a l imited role in mammal ian development and part icipates in the cellular differentiation of only the few cell types shown in Fig. 1. The fusion of gametes to form a zygote is perhaps the most familiar example of developmenta l ly induced cell fusion (Fig. 1A). After using the catabolic activity of the acrosome and burrowing through the zona pel lucida of the oocyte, the sperm fuses wi th a ma tu re egg to p r o d u c e a d i p l o i d zygote (5).

342 Sullivan and Eggan

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The Potential of Cell Fusion for Human Therapy 343

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Fig. 2.Terminology of cell fusion.There are several possible products from cell fusion events. (A) If fusion occurs between two cells of the same type, the resultant membrane-enclosed cytoplasmic body is called a "homokaryon." If the contents of the two nuclei merge to form a single tetraploid nucleus, the result is termed "homotypic synkaryon." (B) If two different types of cells merge, this results in a "heterokaryon" and if the components of two nuclei merge to form a single nucleus, the fusion product is termed a "heterotypic synkaryon." The term "hybrid" in this context refers to the fusion product of any two cells, irrespective of how many nuclei they possess. (C) Also seen during eukaryotic development are syncitia.These are the result of fusion of many cells. Fusagenicity (fusability) is the cell's ability to fuse to other cells. A trigger for fusion, like a chemical or virus, is called a "fusagen."

During placental development , t rophoblast cells fuse to produce a large aggregate of cells called a t rophosyncyt ium (Fig. 2 explains the term "syncyt ium" as well as other terms pertain- ing to cell fusion). Trophoblast cells init ially appear 3-4 d after ferti l ization as the outermost layer of cells in the human blas- t o c y s t (Fig. 1B). A f t e r the b l a s t o c y s t a t t a c h e s to the endometr ium, these cells fuse to form the t rophosyncyt ium, a structure that has a surface area of up to 10 m 2 (6). This large surface area of contact between mother and fetus allows for a more rapid t ransport of nutrients, hormones, and wastes than would be possible with individual trophoblast cells (7). Another example of cell fusion augment ing cell function is the increased contractile capaci ty of muscle enabled by the format ion of myosynci t ia dur ing skeletal muscle deve lopment (Fig. 1C). The myosyncitia, formed from mononucleated myoblasts, have al igned cytoskeletal elements that a l low a higher constriction than unfused myoblasts (8). In at least one case, cell fusion has also been reported to alter cell function completely; osteoblasts, cells that synthesize and mineral ize the collagenous, extracel- lular matr ix of bone can also fuse with macrophage-l ike cells to form osteoclasts, mul t inucleated cells that catabolize and reabsorb bone tissue (Fig. 1D).

Di- and tr inucleated hepatocytes are commonly found in the liver, even under normal conditions. Al though it has been observed that hepatocytes fuse with other cells following tissue t ransplant (9), it has yet to be shown that cell fusion is the under lying mechanism for generating these multinucleate liver cells under steady-state condit ions (Fig. 1E). It is possible that these cells could also arise from a lack of cytokenesis after nuclear division (10).

Cell fusion m a y also p l ay a role in the innate i m m u n e response. When macrophages are exposed to a higher titer of pathogens than they can readi ly engulf and destroy, they fuse together to form cells named "foreign body giant cells." It is not known, however, whether this is a directed process to min- imize the spread of pathogens or is instead merely the result of a b reakdown in the regula tory mechanisms governing cell fusion under such overwhelming condit ions (Fig. 1F; 11).

Even though in lower eukaryotes, especial ly C. elegans (12) and D. melonagaster (13), the molecular and cellular mechanisms underpinning cell fusion are becoming better unders tood (14), it has been a challenge to isolate the proteins that catalyze the fusion of mammal ian cell membranes. Many proteins have been proposed to p lay pivotal roles in cell fusion, but it has

Fig. I. (Opposite page) Mammalian cell fusion occurs spontaneously in vivo. Instances of cell fusion that occur during normal mammalian development: (A) Fe r t i l i za t i on :The haploid sperm fuses with the haploid oocyte forming a diploid zygote.(B) P lacenta l deve lopment :Trophob las t fuse to form the syncytiotrophoblast, allowing faster transport of nutrients, hormones, and wastes across the maternal/fetal boundary. (C) Skeletal muscle fo rmat ion : Myoblasts fuse to form myotubes and myofibers, which have increased ability to contract. (D) Bone fo rmat ion : Bone- building cells called osteoblasts fuse to form bone-resorbing multinucleated osteoclasts. (1=) Liver development: Bi- and trinucleated hepatocytes are commonly found in mammalian cells suggesting cell fusion. (F) Immune response: When macrophages cannot eliminate intracellular pathogens, they fuse to form giant cells that are surrounded by activated T cells.


344 Stdlivan and E ggan

been difficult to separate those that are impor tant in processes in'~xlediat ely preceding fusion (i.e., differentiation, migration, recognition, and adhesion) f rom those impor tant for membrane fusion itself (i5).

C o m m o n l y used approaches for iden t i fy ing molecules involved in cell fusion have included the characterization of antigens bound by ant ibodies that inhibit cell fusion and the characterization of mutants that are defective in cell fusion (4,15). For example, CD0, a member of the te t raspanin protein family has been shown to be required for mouse s p e r m / e g g fusion. Ant i -CD0 a n t i b o d y was in i t ia l ly found to inhibi t sperm-oocyte b inding and fusion in vitro in a dose-dependent manner (16). Later ge ne-targeting ex periment s showed that spe rm b inds normal ly to eggs lacking CD0, but that these mutant eggs could not undergo spe rm-egg membrane fusion (i7). How this part icular protein is important to the fusion mechanism remains unclear, a l though it is hypothes ized that it s tabil izes adhes ion molecules and leads to close contact between egg and spe rm membranes (17).

Recent ly , it has been d i s c o v e r e d tha t o s t e o b l a s t and macrophage fusion share a con'~non regulator: a plasma membrane-embedded protein called DC-specific t ransmembrane domain protein (STAMP). In mice deficient for this protein, both osteoclast and foreign-body giant-cell formation are inhib- ited. When the D CS TAMP gene wasvira l ly reintroduced, fusion of both cell types ensued as it does in cells isolated from wild- type mice (i8). The DC-STAMP protein, initially identified by a DNA subtractive screen between mult inucleated osteoclasts and mononucleated macrophages, is necessary but not sufficient for the induction of cell fusion (i8).

Molecules that have been found to be important for mammalian membrane fusion can be d iv ided into three groups: intracellular proteins (e.g., calmodulin and calpain), membrane proteins (e.g., CD0, vascular cell adhesion molecule [VCAM]-I, ADAM12, Caveolin-3, and DC-STAMP), and secreted factors (e.g., Ca ~+ and interleukin-4; 19). Although all of these m a y b e necessary for fusion of specific cells, none are, by themselves sufficient to induce this process, highlighting the complexi ty of this phenomenon. The importance of certain plasma membrane proteins for cell fusion is i l lustrated by decreased rates of in vitro cell fusion in the presence of proteolytic enzymes like t ryps in (20). In contrast, the use of different catabolic enzymes (e.g., pronase, neuraminidase, or hyaluronidase) can increase fusion between specific cell types (21-23) suggesting that there may be plasma membrane components that inhibit cell fusion or that removing certain proteins destabilizes cell membranes, encouraging membrane fusion.

The biophysical processes that are init iated by cell fusion- inducing molecules are still poor ly unders tood in most cases, but have been the best e lucidated for the generation of muscle f iber synci t ia f rom mononuc lea t e myoblas t s . Init ially, mononucleated cells recognize and adhere to the cells with which they will fuse. These steps are protein dependent and are affected by ambient p H and the presence of ca lc ium (19). Following adhesion, the plasma membranes of two adjacent cells move into close proximity with each other. It has been suggested that this proximity between adjacent membranes is maximized by interactions between proteins on the surface of adjacent cells thereby increasing the probabil i tythat cell fusion will occur (20).

The "stalk-pore" model, a widely accepted theoretical model of cell fusion suggests that ini t ial ly only the outer phospho- lipid layers of adjacent cell membrane bilayers fuse to form an intermediate structure te rmed a "stalk" (15,24). Such struc- tures are unstable but occasionally fusion of the inner l ipid bi layers of the adjacent cells occurs, p roduc ing "pores" or points of cytoplasmic continui ty between the cells (i5). From such pores, merger of adjacent cell membranes continues and vesiculation of the excess membrane around these initial points of fusion occurs (2~27). Al though it is suspected that such complex mult is tep processes are specific and highly regulated, much of this model remains to be exper imental ly verified (15). It is possible that similar steps also take place dur ing the cell fusion events occurring dur ing the differentiation of other cell types or fol lowing cell t ransplanta t ion (28).

Cell Fusion Confusion:Which Cells Undergo Reparative Fusion After Transplant?

Instances of "spontaneous" cell fusion had, unti l recently, thought to be restricted to the examples cited in Fig. 1 and to cells cul tured in vitro. A series of unexpected results arising from t issue t ransplanta t ion and ce 11 culture experiments have demonst ra ted that a review of this model is clearly needed. In a series of experiments carried out over the last several years, t ransplanted neural, hematopoietic, and mesenchymal stem cells all seemed to give rise to deve lopmenta l ly unrela ted tissues in vivo (29). In vitro myocytes seemed to transdifferentiate into adipocytes (30), pancreatic cells into hepatocytes (31), keratinocytes into fibroblasts (32), and endothel ia l cells into c a r d i o m y o c y t e s (33). These o b s e r v a t i o n s were i n i t i a l l y explained by invoking the possibi l i ty that adult s tem cells might transdifferentiate into different cells in response to factors secreted by the environments into which the cells were t ransplanted. However, fol lowing the obser~-ations t hat a vari- ety of s tem cells can fuse with somatic cells in vitro, (34-36), an alternative explanation was suggested: perhaps the cells were merely fusing with other cells and taking on the developmental character of their fusion partner.

Gibson et al. (37) were among the first to capitalize on the realization that many cases of transdifferentiat ion were in fact instances of cell fusion and they hypothesized that cell fusion might also be able to restore cell function to diseased cells. After t ransplanta t ion of dermal fibroblast s into mdx knockout mice, they found that ei ther the fibroblasts themselves or possibly their progeny, fused with the myotube s had par t ia l ly rescued the mice from muscle dysgenesis (37,38). Such rescue was shown to be possible by t ransplant ing wi ld- type hematopoietic cells (39,40). Transplanted hematopoiet ic cells were found to fuse to other cell types in vivo including hepatocytes, car- diac myocytes, Purkinie cells, and ol igodendrocytes , and to change their gene-expression pat terns after fusion (41-43).

Several addi t ional studies show that cell fusion can occur after cellular t ransplanta t ion and that the fusion products are functional, stable heterokaryons. Weimann et al. (44) discov- ered that some of the Purkinje neurons extracted f rom female patients previously treated with male bone marrow cells were tetraploid (XXXY) indicating fusion had occurred between neurons and the t ransplanted cells. In later experiments, where bone marrow cells consti tut ively expressing the fluorescent

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The Potential of Cell Ft~sion for H ~ a n Therapy! 345

marker green fluorescent protein (GFP) where injected into lethally i r radiated wi ld- type mice, GFP posit ive neurons were found in the brain biopsies, again suggest ing cell fusion. Marker analysis showed these cells expressed a neuron-specific marker but no markers of hematopoiet ic s tem cells (H~CCs), suggesting that the neuronal phenotype was dominant in the fusion product . When bone marrow cells that contained a GFP cas- sette driven by a neuron-specific promoter were injected, GFP posit ive cells were found in the cerebellum, again indicat ing that reprogramming of the bone marrow cells had taken place.

Alva rez -Dolado et al. (45) generated t ransgenic mice in which a LacZ reporter gene was placed under control of a tran- scriptional te rminator flanked by loxP sites. When exposed to the Cre protein, this LoxP flanked "stop" element could be excised and the LacZ t ransgene was expressed. In this study, Cre-posit ive bone marrow cells were injected into mice that carried the inactive LacZ reporter. Several months following injection, the animals were sacrificed and examined for the presence of cells that expressed ~-galactosidase activity. [3- Galactosidase-posi t ive cells were found in the liver, brain, and heart , p r o v i d i n g ev idence that cell fus ion had occur red . Whether generat ion of these stable fusion products occurs normal ly dur ing tissue homeostasis and whether fusion is a natural repair process are interest ing questions that remain largely unanswered.

Perhaps most prominently, t issue repai r with cell fusion has also been shown to rescue liver cell function. Work has now been carried out i ndependen t ly in two labs us ing a mouse mode l for hered i ta ry tyros inemia type I, which harbors muta- t i ons in the f u m a r y l a c e t o a c e t a t e h y d r o l a s e gene Fah. Hered i t a ry tyros inemia type I is a rare au tosomal recessive disorder, affecting mainly the liver, kidney, and per iphera l nerves. The symptoms range f romsevere hepatocel lu lar dysfunction in ear ly infancy to chronic l iver dysfunct ion. When wi ld - type bone marrow cells were t r ansp lan ted into the livers of d iseased mice they were found to fuse wi th the Fah l iver cells and rescue cell funct ion (9,46). A l t h o u g h these results are impressive and demonst ra te that reparat ive cell fusion can be used for t reat ing metabolic disorders in model organisms such as the mouse, several challenges need to be su rmounted for such an approach to be used to treat s imilar d isorders in humans.

Cell Fusion forTherapy: Challenges Al te rna t i ve t he r ap i e s for r e s to r ing organ funct ion are

intensely needed because of a scarcity of donor organs and tissues (47). Perhaps, induced cell fusion can be used to repair cel- lu lar dysfunction and thus be therapeutical ly use ful. Indirect ly, cell fusion has highly assisted the s tudy of development and disease, enabl ing the genera t ion of monoclonal ant ibodies through the fusion of lymphocytes and t ransformed cells to produce immortal ized hybr idomas (48). Directly, cell fusion has been proposed as an alternative form of cancer immunother- apy (49) as well as a cell-based gene-del ivery system for treat- ing muscular dys t rophy (50). However, this process has yet to be implemented effectively as a therapeutic tool in vivo.

Fol lowing the realization that the widespread transdiffer- ent iation of hematopoietic cells followingt ransplant in response to a foreign niche does not occur, it has been speculated that

cell fusion m a y be a more realistic approach for res tor ing function to diseased tissue (51). By inducingthe fusion of donor cells to damaged recipient cells a l ready located and integrated in t he tissue (5i), problems associated with engineering organs and ensuring proper integrat ion of t ransplanted cells may be avoided. As interesting as this potential benefit m a y be, however, many challenges need to be faced when considering how cell fusion might be induced and used for the purpose of rescuing cell dysfunction (Fig. 3).

The first such challenge is to determine how the donor cells should be del ivered to the site of tissue injury. Ferrari et al. (52) t ransplanted bone marrow cells into ~u/x mutant mice and some of these cells or their p rogeny travel led to the muscle and fused with it, restoring its function. However, the mechanisms by which these events occurred in the animal model or would occur fol lowing cell t ransplants into human patients is unknown. The cells that fuse wi th the recipient tissue may be carried there by the blood supp ly along with neutrophils , macrophages, and mast cells, which are normal ly directed to areas of injury or disease as part oft he innate immune response. Whether there is an addi t ional chemotactic migrat ion of recipient cells to damaged tissues is unknown. An addi t ional concern facing cell-fusion the rapy is whether t ransplanted cells fuse wi th a wide var ie ty of cell types in the body and whether there are any pathological consequences of these occurrences. Al though there are several reports showing cell fusion after t ransplantat ion, we do not know how sensitive current assays for cell fusion in vivo are, and how prevalent fusion must be to be detectable. Finally, hybr id cells after t ransp lan ta t ion must be bet ter assessed functionally.

Trans-gendertissue t ransplantat ion in humans initially indi- cated that bone marrow cells rescue cell dysfunction and might regenerate damaged tissue. When male cells were transferred into female patients, functional cells containing a Y-chromosome were found (53~q4), indicat ing that t ransplanted cells had integrated into the recipient in some manner. Authors or iginal ly interpreted their data as evidence that male tissue t ransplants contained s tem cells, which transdifferentiated into the cell types required by the host. However, later s tudies looking in injured and normal tissues showed HSC engraf tment to be surpr is ingly rare (55~56), indicat ing that these stem cells l ikely did not s imply transdifferentiate into nonhematopoiet ic line- ages. These t ransplanta t ion data are, of course, also consistent wi th a model in which t ransplanted bone marrow cells fuse at a very low level with the damaged cells. After s tem cell fusion was shown to occur in vitro (34 36), it was suggested that the bone marrow cells responsible for repair ing the damaged cells through fusion were donor H~CCs (57). More recent evidence, however, suggests that differentiated H~cC progeny, rather than HSCs themselves, take part in this process. Markus Grompe and c o l l e a gue s c o n t e n d tha t f o l l o w i n g t r a n s p l a n t a t i o n , macrophages, not s tem cells, can correct genetic disorders of liver and muscle (425859). Although promis ing results have been obtained in these limited syste ms, spontaneous cell fusion between other cell types is very rare, and if this process is to be implemented for therapeutic benefit more generally, the frequency of heterotypic cell fusion must be increased.

Traditional inducers of cell fusion in vitro (i.e., polyethyl- ene glycol, electrofusion, fu sage nic viruses) have at least three

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346 Sullivan and Eggan

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Fig. 3. Challenges to rescuing cell dysfunction by cell fusion.After donor cells reach the site of disease or injury, they must: ( I ) First recognize and adhere to dysfunctional recipient cells. (2) Fuse to damaged cells specifically forming intertypic hybrid cells. (3) Acquire the phenotype of the recipient cell. (4) Maintain a functional phenotype and epigenetic stability.

disadvantages when considered for therapeutic use: they are nonspecific (fusing cells to many other cell type present), cytotoxic, and /or immunogenic. Therefore, other methods of inducing cell fusion need to be explored. One possibility is to express proteins that induce cell fusion in other eukaryotes (e.g., epithelial fusion failure [EFF]-I from C. elegans) in human cells and see if this is sufficient. However, proteins such as EFF- 1, do not have human homologs, so this approach will be immunogenic and noncell-type specific. Furthermore, physi- cal differences between cell membranes of different species m a y m e a n this a p p r o a c h m a y not i n d u c e cell fus ion . Therapeutically, nonspecific induction of cell fusion will probably do more harm than good. Regulated fusion between specific cell types will be required. The best option will probably be to explore the mechanisms by which cell fusion occurs nat- urally and then seek to enhance it.

Despite the substantial challenges presented, overcoming the limitations of cell fusion may enable the treatment of several genetic diseases (e.g., tyrosinemia, hemophilia, hypercholesterolemia,

muscular dystrophy) and so the approach should be considered. However, little is known about the regulation of phenotype in heterotypic hybrid cells after fusion. It is clear, however, that trans-activating factors are important for the emergent phenotype, whose maintenance is dependent on a supply of transcription factors and regulatory mechanisms that repress as well as activate, specific genes (60). Cell-culture experiments fusing pluripotent stem cells with somatic cells have for the most part only generated hybrid cells with a pluripotent-stem cell phenotype (34,36,61-69).

However, this does not necessarily mean this phenotype is dominant, as culture conditions could prevent hybrid clones exhibiting the somatic phenotype from surviving. Takagi et al. (1983) reported the somatic cell phenotype to be dominant but here aneupluripotent fusion partners were moose embryonal carcinoma cells, which were aneuploid. It may be that the trans- formed state of the EC cells allowed the differentiated hybrids to proliferate, as is the case when B cells are fused with cancer cells to produce hybridomas (70). Consistent with this model,


The Potential of Cell Fusion for Human Therapy 347

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Fig. 4.The potential of cell fusion for human stem cell therapy.Three potential strategies for using cell fusion for therapy: (A) Cell function rescue by fusion: Stem cells or their derivatives, transplanted into the patient, fuse with damaged recipient cells and restore cell fusion. (B) In v i t r o mode l i ng of disease using genet ica l ly ta i lo red embryon i c s tem (ES) cell (i) Somatic nuclear transfer: nuclear material from one nonre- productive cell biopsed from a patient will be fused with an enucleated human oocyte. ES cells will be derived from the reconstructed embryo and these cells should display characteristics of the patient's disease. Differentiating these tailored stem cells into the cell type (s) where pathology is exhibited will serve as in vitro model degenerative diease. (ii) Cell hybridization: biopsied cells are fused and reprogrammed by pluripotent stem cells. Normal chromosome complement could be restored to tetraploid hybrid cells by selective genome loss or reductive division.Another alternative is to use cytoplast to reprogram cells. (C) Transp lan ta t ion of differentiated progeny of genet ica l ly ta i lo red ES cells: Tailored ES cells will be differentiated into cell type specifically needed to replace or assist cell needed by the patient.

when A n d r e w s and Goodfe l l ow (1980) fused m u r i n e EC (PCC4azaR) cells with thymocytes, the resultant hybr id cells

thymocyte antigen H-2K (71). This shows that still expressed k reprogramming to an EC phenotype was not complete (even though these hybr id cells were still p lur ipotent as assessed by teratoma generat ion in SCID mice).

It w o u l d be in teres t ing to s tudy the dominance of the plur ipotent stem cell phenotype vs the somatic phenotype in cell hybr ids without selective pressures and to learn how the phenotypic outcome might be controlled. To this end, presence of serum in the media is, in and by itself, problematic. The presence of unknown factors contained in serum direct the resultant phenotype in ways we cannot predict and serum is also seen to prevent maintenance of epigenetic marks (at least in blastocyst cells in vitro [72]). Fortunately, serum no

longer need s to be used, as chemically defined condit ions have been identif ied in which embryonic stem cells and somatic cells can be cultured (73,74). This could possibly provide a neu- tral culture environment from which phenotypes besides that of the embryonic-stem cell phenotype may emerge. If cell fusion is to be used to rescue damaged cell function, conditions needed to make the recipient cell phenotype dominant in cell hybrids will be needed.

It can be speculated that some diseases may benefit from cell fusion, however, the safety issues regarding the generation of tetraploid, potent ia l ly cancer forming, hybr id cells in the patient need to be seriously considered. It has been stated that hybr id cells formed from human stem cells and somatic cells have no therapeutic potential because they are te traploid (75). Surprisingly, te traploid cells can behave in a fashion that


348 SMlivan and Eggan

is analogous to diploid cells dur ing many developmenta l processes. Viable human infants with a large proportion of tetraploid cells have been born (76,77) and have even survived for up to 15 mo after birth (77,78). However, these cases are exceptionally rare and may be more of a testament to the embryo's and early infant 's ability to compensate for many developmental abnormalities.

The relationship between cell fusion and cancer formation needs to be s tud ied further. Many t umor ceils are more fusagenic than theiruntransformed counterparts (14). However, it remains to be seen if cell fusion leads to transformation and metastasis or vice versa. Indeed, it has still to be established that there is a direct relationship between the two phenomena (14). It has been suggested that fusion of macrophages or lymphocytes can render a tumor malignant and that tetraploidy commonly leads to aneuploidy in fused ceils (51). Recently, however, it was shown in mouse that when bone marrow cells were fused to neoplastic intestinal epithelium, tumors did not form (80). Perhaps, if the resultant phenotype of the hybrid cells were quiescent, though still functional, the risk of transformation and cancer would be reduced. In any case, aging stu dies in animal disease models treated by cell transplant and fusion therapies should help to resolve whether this is a legitimate concern.

Methods to restore normal DNAcomplement to hybrid cells should also be considered. Fusing cytoplasts with damaged ceils has been proposed (80), although current research suggests that nuclear components from the reprogramming cell must be present to obtain the desired result (65). If cytoplasts, devoid of chromatin but with these u n k n o w n reprogramming factors, could be isolated, these could potentially be used to restore cell function. Alternatively, perhaps a tetraploid fusion product could have diploidy restored to it. Conceptually, this could happen by at least two mechanisms: reductive division (9) and selective genome loss (81). Reductive division is a process that might be sin~lar to meiosis, allowing the formation of two diploid ceils from a tetraploid cell.

Experiments carried out byGrompe et al. (9) are consistent with, but fall well short of demonstrating, the notion that mouse hepatocytes undergo reductive division. In their experiments, fewer [3-galactosidase-positive hepatocytes than Fah-positive hepatocytes were observed in the liver after transplant, suggesting that not all fusion products continue t o harbor the LacZ transgene. In addition, after serially t ransplant ing wild-type male bone-marrow-derived hepat ocytes into the livers of irra- tiated female Fah knockout n-dce, approx 33% of male cells in female serially transplanted recipients showed nonfusion kary- otypes (40 XY, rather than 80 XXYY). However, liver ceils fre- quently fuse and divide, and it remains to be seen if these cells are the result of legitimate reductive divis ions and, if so, whether this phenomenon can be observed in other cell types.

The second process by which diploid ceils might be generated after human cell fusion is selective genome loss, a process i n v o M n g the removal of one parent 's genomic DNA from an egg-sperm fusion product. This process is seen in one type of reproduction in which the ovumand spermfuse but the genome originating from one of the two parents is lost. A paper pub- lished last year showed that males of Wasmannia m~rop~mctata (the little fire ant) colonies clone themselves by causing the loss of the maternal genome (81). The mechanism is, as yet,

Stem Cell Reviews

u n k n o w n and it remains to be seen if such a process could be recapitulated in mammal ian cells and harnessed for directing selective genome loss in human fusion products.

Conceptually, cytoplast fusions may be the most desirable route to pursue for therapeutic applications for cell fusion, as mechanisms unde rp inn ing reductive division and selective genome loss remain unelucidated and such complex processes will, most probably, prove extremely difficult to regulate in vivo.

Conclusion The therapeutic use of cell fusion may be underestimated.

It is certainlythe case that many documented instances of stem cell plasticity are in fact the result of cell fusion. Because methods for tracking cell fusion in vivo are laborious and time con- sun-ring, it is still unclear how widespread cell fusion is in human tissues dur ing either development or following cell transplant. There fore, t he importance of cell fusion for human tissue repair and homeostasis is still speculative. However, experiments in animal model systems, including a metabolic liver disease mouse model, where strong selective pressures for fusion products exist, argue that there may be utility to this approach.

Efforts to elucidate mechanisms underp inn ing mammalian cell fusion will continue and it is expected that the current challenges (particularly the low fusion frequencies observed thus far) may be surmounted. A higher unders tanding of how cell fusion is regulated will also assist in the treatment of fusion defects in egg and sperm (leading to infertility), trophoblast ceils (leadingto placenta problems such as preclampsia), muscle cells (leading to myopathy and muscle dystrophy), bone cells (leading to low osteoclast formation frequencies) as well as providing further insight into transmission of viral infec- tion and cancer. Additionally, as cell fusion has been impli- cated in transformat ion and cancer progression (14), more must be learned about controlling the function and phenotype of fusion products.

Acknowledgments SS is funded by a fellowship from the Harvard Stem Cell

Research Fund. We thank Prof. Amy Wagers for helpful dis- cussions regarding content and Renate Helln-dss-Peralta for creating the images. We also thank Kit Rodolfa, David Wells Staudt, Kevin Jwo and Dr. John Dimos for proofreading the manuscript.

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The potential of cell fusion for human therapy

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