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Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system

2012-06-04 17:41:13

Journal of Cell Science; 2012 Jun 15; 125(Pt 12):2837-43


Amy M. Skinner, Markus Grompe and Peter Kurre



Introduction



Non-pathogenic genetic variation and genome-wide copy number variation (CNV) have been demonstrated in somatic tissues of several organisms, including humans, fruit flies and yeast (Conrad et al., 2010; Hastings et al., 2009; Torres et al., 2010). Mechanisms proposed for the generation of CNV implicate cell-intrinsic means of DNA recombination (Zhang et al., 2009). Polyploid progeny of murine hepatocytes undergo ploidy reduction, propagate CNV, and generate genetically diverse events (Duncan et al., 2010). However, a recent study also demonstrates cell fusion between bone-marrow-derived cells (BMDCs) and hepatocytes, suggesting that CNV is generated by merging DNA from two separate cells rather than from recombination events within a single cell (Duncan et al., 2009).



Bone-marrow-derived cells can fuse with hepatocytes, neurons or epithelial cells of the intestine, respectively, in a process that is seemingly amplified by acute tissue damage or inflammation (Bailey et al., 2006; Davies et al., 2009; Johansson et al., 2008; Nygren et al., 2008; Rizvi et al., 2006; Wang et al., 2003; Willenbring et al., 2004). Such products of ‘heterotypic’ cell fusion have been found to acquire functional characteristics of the host tissue and are considered evidence for a physiological regenerative mechanism (Palermo et al., 2009). Other than in the liver, direct tracking of genetic markers in fusion events and serial evaluation of mitotic competence and cell fate have not been extensively performed. This probably reflects the experimental focus of previous studies, technical limitations or the post-mitotic nature of specific fusion partner cell types (e.g. neurons). Aside from rare reports of incidentally detected somatic mosaicism, changes in genomic copy number the hematopoietic system are generally associated with malignant transformation (Piotrowski et al., 2008).



Congenic mice harboring polymorphisms at the Ly5 locus expressing distinct (CD45.1, CD45.2) cell surface markers are frequently used to dissect donor–host contributions for the study of hematopoietic stem cell (HSC) function and cell–cell fusion (McCulloch and Till, 1960; Zebedee et al., 1991). Co-expression of both CD45 donor and host isotype cell surface markers after ablative transplantation is widely attributed to experimental artifact, or considered evidence of membrane protein transfer between hematopoietic cells (Cho and Hill, 2008; Yamanaka et al., 2009). Here, we carefully dissect events with parental marker co-expression and present evidence of hematopoietic ‘homotypic’ cell fusion (i.e. fusion between cells arising in the same tissue) and marker CNV by interphase FISH and SNP-PCR. We observe homotypic hematopoietic fusion at comparable rates under non-injury conditions in a parabiosis model and show that intra-hematopoietic cell fusion produces mitotically competent, clonogenic progenitors that are genotypically diverse for unique informative markers without evidence of malignant transformation.



Results and Discussion



Intra-hematopoietic cell fusion events isolated from irradiated transplant animals



We hypothesized that cell fusion could be a potential mechanism for generating genetic diversity within hematopoietic tissues and sought to identify intra-hematopoietic cell fusion progeny (Anderson et al., 2011; Chandhok and Pellman, 2009). In independent experiments, sublethally irradiated male recipients (CD45.1) received either congenic, CD45 (Ly5)-mismatched c-kit+, sca-1+, Lin- (KSL) cells, or unfractionated bone marrow cells from female donors (CD45.2) transgenic (hemizygous) for human CD46 (Fig. 1A) (Yannoutsos et al., 1996). In an additional model, sublethally irradiated CD45.1 GFP+ males received whole bone marrow from CD45.2 females transgenic for human CD46 (Fig. 1A). Following hematopoietic reconstitution of recipients with multi-lineage donor chimerism (40–90%) in the peripheral blood, the hematopoietic tissues were harvested for analysis at time points between 1 and 12 months after transplantation. Cells co-expressing human CD46 (donor) and either CD45.1 cell surface antigen or GFP (both host) were serially sorted for improved stringency (Fig. 1B). A ‘doublet discriminator’ was used to exclude isolation of ‘doublets’ (i.e. two attached cells) (Hughes et al., 2009; Wersto et al., 2001). We observed cells co-expressing parental markers in all donor–host combinations and following different FACS sorting strategies (Fig. 1B). Shared marker expression in individual, sorted cells was confirmed by immunofluorescent (IF) deconvolution microscopy and z-stack analysis (Fig. 1C,D), distinguishing cells co-expressing donor–host markers from doublets. To exclude ambiguity from surface antigen or membrane transfer between donor and host hematopoietic cells (Yamanaka et al., 2009), DNA evidence of fusion was demonstrated by single nucleotide polymorphism (SNP) typing (D1Mit421.1) for CD45.1 and CD45.2 alleles. Genomic DNA from flow-cytometrically isolated single CD45.1+ CD46+ cells revealed amplification of both donor and host SNPs (Fig. 1E), whereas cells sorted from control animals demonstrated only their predicted unique donor or host CD45 SNP signature (supplementary material Fig. S1). Validation of cell fusion within the sorted population was further corroborated by interphase fluorescence in situ hybridization (FISH) analysis (Fig. 1F) showing synkarya containing genetic markers of both donor (human CD46) and host (mouse Y chromosome) origin. These observations suggest the acquisition and expression of genetic markers through fusion between hematopoietic cells and illustrate their long-term persistence.



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