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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0962fje. |
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,||,¶,¶,2
* Transplantation Biology,
Department of Physiology,
Department of Internal Medicine,
Department of Surgery,
|| Department of Immunology, and
¶ Department of Pediatrics, Mayo Clinic, Rochester, Minnesota, USA
2Correspondence: Mayo Clinic, Transplantation Biology, 200 First St. SW, Medical Sciences 2-66, Rochester, MN 55905, USA. E-mail: platt.jeffrey{at}mayo.edu
SPECIFIC AIM
Spontaneous fusion of hematopoietic stem cells with parenchymal cells has recently been observed in certain instances of tissue injury and disease. We asked whether spontaneous fusion could occur under "physiologic" conditions and, if so, whether fused cells could contribute to the normal body plan and whether cell fusion could underlie transfer of retroviruses.
PRINCIPAL FINDINGS
1. Human cells can spontaneously fuse with porcine cells in vivo
In the course of studying human hematopoietic stem cell engraftment in swine, we asked whether human cells fuse with porcine cells. To address that question, we studied swine in which human hematopoietic stem cells had been injected in utero. Piglets so injected were found to have human cells in the circulation and tissues for more than one year. Peripheral blood leucocytes from piglets with established chimerism for up to one year were incubated with Epstein-Barr Virus (EBV). EBV efficiently immortalizes human B cells, but not porcine B cells because porcine B cells lack the receptor necessary for viral entry, thus enabling selective expansion of human cells. Our own controls confirmed this specificity (data not shown). Metaphase spreads from EBV-transformed cells generated from the peripheral blood of chimeric pigs were analyzed by g banding. Nuclei of the immortalized cells contained only 43 ± 1 total chromosomes (Fig. 1
A) and as expected, human diploid cells had 46 and porcine cells had 38. In addition, the chromosomes had a mixed banding pattern that varied from cell isolate to cell isolate and between 6 piglets tested suggesting that the immortalized cells contained DNA from both species.
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To determine if immortalized cells contained DNA from both species, we probed the DNA for a porcine-specific repeat sequence as well as for the human-specific Alu sequence used for initial cell screening by in situ hybridization. One hundred percent of immortalized cells were positive for Alu, and nearly 95% of immortalized cells were positive for the porcine repeat sequence (Fig. 1B
). Thus 95% of cells were positive for both Alu and the porcine repeat sequence and contained both human and swine DNA. The hybrid cells expressed a mixture of human and porcine proteins. The results suggest that synkaryons formed as a result of cell fusion.
2. Human-porcine hybrids contribute to porcine epithelial tissues
Concomitant maintenance of human and porcine DNA and simultaneous expression of human and porcine cell surface proteins by hybrid cells indicates a fusion-induced alteration in phenotype; we were further interested in the possibility of altered cell function as a result of cell fusion. We therefore asked whether human cell hybrids had acquired phenotypic properties of their fusion partner and could thus contribute to the porcine body plan. To this end, we performed in situ hybridization for Alu in tissues obtained from organs of chimeric swine. Alu positive cells were found in epithelium of kidney and skin, albeit rarely. The location and distribution of the Alu positive cells were consistent with the possibility that one cell entered the tissue and gave rise to a line of cells that contributed to non-hematopoietic porcine tissue structures.
Human cells found in the tissues could have arisen either from human hematopoietic stem cells that had undergone transdifferentiation autonomously or from fusion of human hematopoietic stem cells with differentiated porcine cells. To distinguish between these possibilities, we studied histologic sections prepared from kidneys of chimeric pigs stained with anti-Gal
1,3Gal antibody and probed by in situ hybridization for Alu. We found Alu+/Gal1,3Gal+ cells (
60% of Alu+ cells found) located in epithelial structures, supporting the notion that cell fusion gives rise to developmental plasticity (i.e., transdifferentiation) and therefore the ability to contribute to the body plan. We also found 40% of the Alu+ cells in the kidney that were Gal1,3Gal-, which supports the possibility that human cells had the capacity to transdifferentiate autonomously in the pig.
3. Human-porcine hybrids contain PERV polymerase gene
Of perhaps more importance than cell fusion-induced transdifferentiation is the possibility that cell fusion could serve as a route for viral transmission. To investigate this possibility, we probed immortalized cell hybrids for porcine endogenous retrovirus (PERV) DNA. PERV was sought in hybrids by real time PCR (Fig. 2
A) and standard PCR (Fig. 2B
) and by in situ hybridization (Fig. 2C
). Real time PCR analysis was conducted on hybrid cells that had been cytospun and stained via in situ hybridization for Alu. Cells positive for Alu were removed from the slides by laser capture microdissection to avoid contamination by porcine cells. Hybrid cells contained substantial levels of PERV pol gene (2.5x104±1.4x104), albeit statistically less than porcine controls (6.6x104±1.8x103). Our findings raise the possibility that infection of human cells by PERV as previously reported occurred through an intermediary step, the formation of cell hybrids.
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To determine whether hybrid cells were capable of infecting human cells with PERV, we co-cultured the hybrid cells with human kidney fibroblasts (293 cells). We used a procedure described previously in which a porcine kidney cell line (PK15) was found to infect human cell lines including 293 cells. The 293 cells we used were passaged every 3 days after co-cultivation with the hybrid cells. Fourteen days later (after 4 passages), 293 cells were separated and analyzed for PERV DNA by real time PCR as before. The 293 cells contained PERV DNA (2.7x102±1.2x102 copies; Fig. 2A
). Thus human cells that had undergone spontaneous cell fusion with porcine cells contained porcine endogenous retroviruses and were capable of transferring that virus to healthy human cells.
CONCLUSION AND SIGNIFICANCE
Here we report that human hematopoietic cells, possibly stem cells, can fuse spontaneously with porcine cells in vivo and that the hybrid progeny contain genotypic and phenotypic elements of each fusion partner. The hybrids are synkaryons with a 2N ploidy value. The synkaryon hybrids have the capacity to contribute to the porcine body plan perhaps owing to extensive cell division. The hybrids contain the porcine endogenous retrovirus and are able to transmit the virus to uninfected human cells (Fig. 3
).
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Approximately 60% of human cells surviving long-term in the pig were cell hybrids. The sustained survival of human hybrid cells over their unfused counterparts suggests that cell fusion may offer a competitive advantage for survival of human cells in the pig. Presumably the advantage is conferred through the acquisition and expression of essential and/or more compatible genes. For example, a functional 1,3 galactosyltransferase gene was present in all hybrids tested, suggesting that production of Gal1,3Gal might promote survival in the pig. Perhaps the presence of Gal1,3Gal enhances the interaction of human cells with other porcine cells and porcine cell products.
Cell hybrids were located in epithelial tissues and were therefore capable of contributing to the body plan. Our data support the idea that hematopoietic precursors in the bone marrow fused with porcine cells underwent transdifferentiation to contribute to non-hematopoietic tissues. Acquisition of broad developmental potential by a cell relatively committed to a specific lineage (or transdifferentiation) is thought to be cell autonomous. Recently, however, fusion has been offered as an alternate means of transdifferentiation. The studies described here offer further evidence that stem cells may utilize the "machinery" of the fusion partner to alter developmental fate.
Cell fusion and in particular the formation of synkaryons may explain viral transfer across species. Here we show that hybrid cells contain PERV DNA. Acquisition of PERV DNA presumably occurred by fusion, although it is conceivable that instead fusion gave rise to conditions that facilitated infection. Although we directly introduced human cells into pigs, it is not beyond imagination that cells might be exchanged between species in the course of routine interactions, contact with insect vectors, parasitism, or when human cells or tumor tissues are implanted in animals. If such exchanges led to cell fusion and formation of synkaryons, it could lead to genetic recombination to yield novel viruses that can infect normal recipient cells.
Thus, transdifferentiation and retroviral transmission are two biological consequences of interspecies cell fusion. Given these results, understanding the mechanisms of cell fusion could have enormous benefit both to devise novel therapeutics for disease states and to avert the spread of disease.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0962fje; 
This article has been cited by other articles:
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  G. C. Schatteman, M. Dunnwald, and C. Jiao Biology of bone marrow-derived endothelial cell precursors Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H1 - H18. [Abstract] [Full Text] [PDF]
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 M.-A. von Mach, J. G. Hengstler, M. Brulport, M. Eberhardt, W. Schormann, M. Hermes, D. Prawitt, B. Zabel, J. Grosche, A. Reichenbach, et al. In Vitro Cultured Islet-Derived Progenitor Cells of Human Origin Express Human Albumin in Severe Combined Immunodeficiency Mouse Liver In Vivo Stem Cells, December 1, 2004; 22(7): 1134 - 1141. [Abstract] [Full Text] [PDF]
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