Figure 5 Laser Capture Microdissection, Exome Sequencing, and Whole-Genome Sequencing of Single Adipocytes Isolated from Study Subject 1 Show full caption (A) Representative photomicrographs of individual mature fat cells isolated from study subject 1 prior to capture. Triglycerides were stained with BODIPY and nuclei with Hoescht. Cells were isolated using laser capture microdissection, their genomes amplified using whole-genome amplification, and exomes and genomes were sequenced in order to determine their genotypes. The individual cells are cell #2, cell #10, and cell #16, and their corresponding genotypes, determined by exome and whole-genome sequencing, are shown in (B) and (C), respectively. Scale bar is 100 μm. (B) Exome-wide distribution of donor- and recipient-specific homozygous SNPs in single adipocytes isolated from study subject 1. Only samples with five or more variants passing the filters were included. Each row represents a sample, and each column represents a SNP position; the top two rows represent donor (pink) and recipient (blue) whole-blood gDNA while the rows below represent analyses of gDNA from single cells. All homozygous SNPs that differ between the donor and recipient and from which at least one single cell displays a signal above the defined threshold are depicted. Each detected SNP in the single cells has been assigned a color depending on whether the genotype at that position corresponds to the donor (pink), the recipient (blue), or both (yellow). Positions where no signals above the defined thresholds were detected are depicted in gray. Out of 27 analyzed cells from this subject, cell #10 displayed mixed genotypes, cell #16 displayed donor-specific genotypes, and the remaining cells, represented here by cell #2, displayed recipient-specific genotypes. Further descriptions are found in the Supplemental Information and Figures S4 and S5 (C) The classification of cell origin of cell #2, cell #10, and cell #16 determined by exome sequencing was confirmed by whole-genome sequencing. Circle plots detail the number of detected donor, recipient, and mixed variants across the entire genome identified in these three cells.

To establish whether the presence of donor-derived DNA in recipient fat cells could be due to differentiation or cell fusion, we developed a technique to directly assess the genotype of single adipocytes (for details, see Supplemental Experimental Procedures ). Purified adipocyte fractions from three BM- or PBSC-transplanted recipients were embedded in agarose and mounted on membranes used for laser capture microdissection (LCM) ( Figure 5 A). Single adipocytes were subsequently isolated, and genomic DNA (gDNA) from each cell was subjected to whole-genome amplification (WGA). Next, sequence libraries were prepared followed by exome capture and massive parallel sequencing of samples from single adipocytes and whole-blood gDNA isolated from both the donor and the recipient. To distinguish between donor and recipient DNA, homozygous SNPs unique for either donor or recipient were initially identified by comparing exome sequences of whole-blood gDNA. These positions were then called in the single-cell exome data (where signals above the defined threshold could be detected) as either donor, recipient, or mixed genotypes ( Figure 5 B and Figures S4 and S5 ). As shown in Table S2 and Figure S4 , only samples displaying five or more variants passing the filters were included in the analysis. In study subject 1 and 2, data from 27 and 24 cells, respectively, fulfilled this criterion, while in study subject 3, data from only 15 cells passed the filters. Consistent with the findings from the bulk adipocyte analysis, the majority (∼90%) of the adipocytes contained SNPs representing patient-derived DNA. Two cells, #4 from study subject 1 and #10 from study subject 2, displayed a single donor-derived variant while otherwise displaying entirely recipient-derived genotypes (90.9% of 11 variants and 97.8% of 45 variants, respectively) ( Figure S4 ). As explained in the Supplemental Experimental Procedures , these rare and unexpected donor genotypes may be the result of artifacts arising from sample preparation and/or erroneous genotype calls in the data analysis. From study subject 3, all analyzed cells displayed recipient-specific genotypes ( Figure S4 ). Cell #10 in study subject 1 displayed mixed genotypes from both the donor and the recipient (based on a total of 42 variants) ( Figure 5 B and Figure S4 ). This is consistent with previous studies demonstrating cell fusion between BM-derived cells and other non-hematopoietic cell types. Similarly, in study subject 2, one cell (#11) contained both donor and recipient variants, which could indicate a cell fusion event ( Figure S4 ). However, this cell displayed only five variants and thus only weakly passed the filters established for classifying whether a cell is of donor, recipient, or mixed origin. Data interpretation for this cell must therefore be considered less confident than for cell #10 from study subject 1. Two adipocytes (morphologically indistinguishable from recipient-derived fat cells), #16 from study subject 1 ( Figures 5 A and 5B and Figure S4 ) and #24 from study subject 2 ( Figure S4 ), harbored donor-specific genotypes, indicating that circulating BM/PBSC-derived progenitor cells can differentiate into adipocytes in humans. Finally, we selected three previously exome-sequenced samples from donor 1 for whole-genome sequencing, which included one recipient-derived cell (cell #2), the donor-derived cell (cell #16), and the cell of mixed origin (cell #10) ( Figure 5 C). This allowed us to analyze recipient- and donor-specific variants across the entire genome and confirmed the conclusions drawn from exome sequencing regarding the origins of these cells. Thus, both exome and whole-genome sequencing at the single-cell level suggest that, in transplanted subjects, BM/PBSC-derived progenitors incorporate into WAT via at least two possible and separate mechanisms, namely adipocyte differentiation or cell fusion. Admittedly, we have no direct proof for either process, as this would require us to follow the fate of single cells in vivo, techniques that are clearly not available for studies in man.