Progress Update: Single Cell Transcriptomics

Progress update by Dr. Pakula

“Understanding initiation and progression of FSHD-1 disease utilizing novel Single Cell Transcriptomics Technology” (April 2018- November 2018)

The goal of our studies is to characterize single DUX4 expressing cells to understand the mechanism of disease initiation and progression. Elucidating an exact mechanism of disease initiation (DUX4 expression) and further progression (consequences of DUX4 expression)- might help us to find biomarkers that will indicate at the exact disease stage and might thus lead to the more precise diagnostics which in the long-term perspective should give the physicians a better idea of the treatment needed at the particular moment of disease development. We also aim to compare FSHD-1 vs matched control cells from unaffected first-degree relatives to understand in more details which molecular pathways are involved in disease development. Discovering them, by using this technology, should give researchers working on FSHD a deeper insight into designing future, targeted treatments.

Before proceeding with the single-cell sequencing we wanted to make sure that selected primary FSHD-1 lines express DUX4. To do that, we have performed immunostaining and calculated the number of DUX4 (+) cells per 1000 cells (see Figure 1).

FSHD-1 CellFSHD-1 Cell

Cell lines

12A del

20A del

09A bic

17A bic

Number of DUX4 (+) cells





Figure 1. Immunostaining of DUX4 (+) cells from FSHD-1 cell lines (DUX4 is expressed in nuclei indicated by arrow). Left: 4x; Right: 40x. Table below images, represents number of DUX4 (+) cells found per 1000 cells on immunostaining at day 4 in differentiation medium. The same batch of cells was delivered to the Single Cell Transcriptomics facility. (Bic-biceps; del-deltoid; A- FSHD-1 subjects)

Once DUX4 expression has been confirmed by immunostaining, cells were subjected to single-cell capturing in hydrogels using the inDrops platform. Patient cells (A) and matched control cells from unaffected first-degree relatives (U) were processed on the same day to avoid batch difference: 17Abic (2x), 17UBic, 09Abic, 09Ubic on day 1; and 20Udel, 20Adel (2x), 12Adel, 12Udel on day 2. Approximately 9000 cells were collected per individual, from which ~6000 cells were sequenced and the remainder kept as backup cells. After capturing cells in hydrogels with inDrops, libraries were prepared and sequenced at the Dana-Farber Cancer Institute.

Because not every transcript present in a cell is sequenced, the fraction of cells in which DUX4 is detected may be less than indicated in Figure 1, a phenomenon known as “dropout”. We also do not exclude that some of DUX4 cells might have died during the hydrogel capture, since they are more susceptible to stress. In a preliminary analysis of the inDrops data, we detected DUX4 itself in only 4 out of ~34000 FSHD cells sequenced. However, DUX4 expression might be also inferred based on elevated levels of DUX4 target genes. By using this approach, we have identified 42 of the ~34000 FSHD cells in which at least one of these three target genes is detected: ZSCAN4, TRIM43, RFPL4B. Characterization of our data is still in progress. We aim to characterize the 42 DUX4-expressing cells by finding their unique signature compared to non-DUX4-expressing cells. We also aim to group these selected DUX4-expressing cells based on the levels of DUX4 target genes, and correlate them with early and/or late apoptosis markers to be able to select “early” and “late” stages of DUX4 activation.

See grant Understanding the initiation and progression of FSHD-1 disease by use of the novel technology called single cell transcriptomics.