Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity

To the editor:

An understanding of hematopoietic stem cell (HSC) biology is therapeutically important because of the many human diseases that result from errors in HSC regulation or that can be treated with HSC transplantation. The zebrafish is a powerful model for studying HSC biology in vivo^1-3 ; however, monoclonal antibodies are challenging to generate for use with zebrafish hematopoietic cells.⁴  Here, we report the development and characterization of a monoclonal antibody to zebrafish Cd41 and show that it can be used to negatively enrich for HSCs.

Zebrafish Cd41, also known as GpIIb and Itga2b, is a cell-surface protein expressed at high levels on thrombocytes and at lower levels on HSCs.^5-7  In humans and other species, CD41 forms cell-surface heterodimers with CD61, also known as GPIIIa or ITGB3. The CD41/CD61 (GPIIb/IIIa) heterodimer plays a critical role in platelet activation, and deficiencies in its function cause the human bleeding disorder Glanzmann thrombasthenia.^8,9  Previous work has shown that Tg(cd41:EGFP) zebrafish can be used to positively enrich for HSCs.^6,10  In adult zebrafish, cd41:EGFP^Low cells in kidney marrow have HSC activity, whereas cd41:EGFP^High cells in peripheral blood are thrombocytes.⁶  These data suggested that an antibody to Cd41 could be useful for studying the zebrafish thrombocyte lineage and for isolating zebrafish HSCs.

To generate a monoclonal antibody to zebrafish Cd41, in 293T cells we expressed a secreted form of zebrafish Cd41/mouse CD61 in which the transmembrane domains of each protein were replaced by an acid peptide and base peptide, respectively, to promote leucine zipper-mediated heterodimerization and secretion. The heterodimeric protein was then used as an immunogen in the production of mouse hybridomas (see supplemental Figure 1A, available on the Blood Web site). Hybridomas were screened using a cell line expressing native zebrafish Cd41 and zebrafish Cd61b. The Cd61b was FLAG-tagged to allow confirmation of heterodimer expression on the cell surface without altering the Cd41 epitope. This yielded a reactive hybridoma that was selected for further subcloning and characterization (supplemental Figure 1B).

We initially tested the Cd41 antibody by flow cytometry using peripheral blood from adult Tg(cd41:EGFP) zebrafish. This revealed that the Cd41 antibody recognizes cd41:EGFP-positive cells with greater than 99% sensitivity and specificity (Figure 1A). Based on light scatter characteristics, these cells are predominantly located in the expected region,⁵  which has traditionally been termed the “lymphoid” gate (Figure 1B).¹¹  At least 2 discrete populations of double-positive cells are discernable; the most abundant of these is relatively restricted to the lymphoid gate. Cytopreparations of cells sorted by flow cytometry demonstrated that Cd41 antibody-positive cells are predominantly thrombocytes, as anticipated (Figure 1C). Furthermore, RNA sequencing confirmed that the most highly expressed genes in antibody-positive cells/zebrafish thrombocytes are well known to be expressed by human and mouse platelets and that there is significant evolutionary conservation among the 3 species (supplemental Tables 1 and 2; supplemental Figure 2). As an alternative method to flow cytometry, the characteristics of the Cd41 antibody were assessed by immunofluorescence (supplemental Figure 3); congruent with the flow cytometry results, the Cd41 antibody stained cells in immunofluorescence experiments with greater than 95% sensitivity and specificity.

To confirm that the Cd41 antibody recognizes zebrafish Cd41, we tested the antibody against a cd41 mutant zebrafish (itga2b^sa10134) that is expected to lack expression of Cd41.¹²  Homozygous mutant zebrafish are observed at much less than the expected frequency in heterozygous crosses but have circulating thrombocytes. Analysis of peripheral blood from cd41^−/− zebrafish by flow cytometry revealed a loss of Cd41 antibody staining (Figure 1D). The proportion of peripheral blood cells stained was normal in heterozygous mutant zebrafish (Figure 1D), but the median fluorescent intensity of heterozygous cells was two-thirds that of the wild-type cells (Figure 1E). This is consistent with decreased cell-surface expression of Cd41 in heterozygous cells. Because loss of Cd41 at the cell surface might compromise Cd61b cell-surface expression, we confirmed that the Cd41 antibody binds to Cd41 and not Cd61b in the Cd41/Cd61b heterodimer using adult Tg(cd61b:EGFP) zebrafish (Figure 1F).

Having characterized the Cd41 antibody for use with adult zebrafish peripheral blood, we turned our attention to adult zebrafish kidney marrow. In kidney marrow, the Cd41 antibody identifies a subset of cd41:EGFP-positive cells, specifically the population with higher cd41:EGFP expression (Figure 2A). Multiple possibilities for this differential binding exist, including transgene activation in the absence of transcription at the endogenous locus and production of discrete epitopes in the 2 cell groups. Because some Cd41 antibody-positive cells isolated from kidney marrow in the absence of a significant forward scatter size limit appeared to be immature when examined using cytopreparations, and because a minority are located within the so-called “precursor” gate, we performed colony-forming assays to determine if some of these cells were progenitor cells that had not yet terminally differentiated. This revealed that Cd41 antibody-positive cells exhibit colony-forming activity comparable to what is obtained with cd41:EGFP^{Low/Very Low}-positive cells (Figure 2B). In this case, the lower level of colony-forming activity in the Cd41 antibody-negative cell population reflects the gating strategy, which includes all Cd41 antibody-negative cells: although HSCs and progenitors such as common myeloid progenitors, common lymphoid progenitors, and granulocyte-monocyte progenitors would be included, abundant terminally differentiated cells such as mature myeloid cells, lymphoid cells, and red blood cells are also included. As expected, cd41:EGFP^High cells do not form significant numbers of colonies because they are largely mature thrombocytes.^5,6 

Because the cd41:EGFP^High population is enriched for thrombocytes and the cd41:EGFP^Low population is enriched for HSCs,⁶  we sought to determine whether the Cd41 antibody permits enrichment of adult zebrafish HSCs by negative selection. We performed limiting dilution transplantation experiments using kidney marrow from Tg(cd41:EGFP;ubi:mCherry) zebrafish that was sorted into a cd41:EGFP^Low, Cd41 antibody-positive fraction and a cd41:EGFP^Low, Cd41 antibody-negative fraction. The sorting strategy included a restriction on forward scatter and used splenocytes to help more rigorously define the cd41:EGFP^High population (supplemental Figure 4A). Morphologically, the Cd41 antibody-positive fraction contained thrombocytes (supplemental Figure 4B) and the Cd41 antibody-negative fraction contained cells that were mostly immature with high nuclear to cytoplasmic ratios (supplemental Figure 4C). These fractions were transplanted into irradiated recipients and engraftment was assessed at 3 months posttransplant. Engraftment was never obtained from cd41:EGFP^Low, Cd41 antibody-positive fractions; by contrast, engraftment was frequently obtained from cd41:EGFP^Low, Cd41 antibody-negative fractions (Figure 2C). Engraftment was multilineage based on expression of the ubi:mCherry transgene in erythroid, myeloid, precursor, and lymphoid gates by flow cytometry (Figure 2D).

The Cd41 antibody characterized in this study is the first monoclonal antibody to permit HSC enrichment by flow cytometry in zebrafish. Limiting dilution software suggests that the frequency of HSCs in our cd41:EGFP^Low, Cd41 antibody-negative fraction is 1/456 (302-688) but this is likely an underestimate resulting at least in part from transplantation being performed without matching major histocompatibility loci between donors and recipients. Our results support the broad use of the Cd41 antibody with other transgenic zebrafish lines in the quest to purify zebrafish HSCs. Indeed, the Cd41 antibody represents a first step toward developing a set of zebrafish “lineage-negative” markers that permit the enrichment of HSCs, as is already done in mice and humans.