By studying the relationships of these subpopulations to

By studying the relationships of these subpopulations to each other, it was found that GMPs are able to create neutrophils but unexpectedly lack the potential to form eosinophils and basophils. Furthermore, and against the prevailing assumption, the GMPs were found to be derivatives of the same branch of hematopoiesis as the lymphocytes, pointing toward altered lineage relationships in human hematopoiesis (Görgens et al., 2013b). Accordingly, we recently proposed a revised model of human hematopoiesis (Görgens et al., 2013a, 2013b). Another outcome of this study was the observation that under the conditions used, MPPs cannot self-renew in vitro; following their first in vitro cell division, they apparently create CD133-positive LMPPs and CD133-negative EMPs, maybe by means of ACD (Görgens et al., 2013a, 2013b). Enforcing assumed roles of ACDs in this lineage-separation process, asymmetric segregation of CD133 molecules was observed in a proportion of dividing CD34+ cells at the intracellular level (Fonseca et al., 2008). In contrast, and independent of its intracellular distribution, the extracellular component of CD133 appeared to be symmetrically distributed on all dividing CD34+ cells (Beckmann et al., 2007; Fonseca et al., 2008). In addition to the cell-fate analyses and ACD studies, we compared the distribution of CD133 at the subcellular level on freshly isolated and cultured HSPCs. Upon cultivation, HSPCs adopt a polarized morphology, forming a leading edge at the front and a leukocyte-specific structure, the uropod, at the rear (Giebel et al., 2004; Rajendran et al., 2009). While CD133 showed a rather random appearance on freshly isolated HSPCs, it redistributes to the uropod tips in cultured HSPCs (Giebel et al., 2004; Görgens et al., 2012). In our studies, we learned that the CD133 epitopes that are recognized by commonly used anti-CD133 EPI-001 (AC133 and AC141) are sensitive to paraformaldehyde fixation (Giebel et al., 2004). Both of these antibodies have been reported to recognize spatially distinct, glycosylation-dependent residues on the extracellular CD133 loops (Bidlingmaier et al., 2008; Miraglia et al., 1997). Recently, a novel monoclonal anti-CD133 antibody, the HC7 antibody, has been described, which specifically recognizes glycosylation-independent protein residues of both extracellular loops of CD133 (Swaminathan et al., 2010). This antibody had been successfully used in a variety of applications including western blot, immunofluorescence, flow cytometry, and immunohistochemistry using cancer cell lines (Swaminathan et al., 2010). To test for the usefulness of the HC7 antibody in hematopoietic stem cell research, we comprehensively compared the presence and subcellular distribution of HC7 and glycosylation-dependent CD133 epitopes on freshly isolated and cultured UCB-derived CD133+CD34+ HSPCs. Furthermore, we tested whether binding of the HC7 or the AC133 antibody exerts any functional impact on the biology of human HSPCs, including their potential to home and engraft into immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After showing that neither AC133 nor HC7 antibodies recognizably alter biological features of human CD133+CD34+ cells and that both antibodies allow detection of living, asymmetrically dividing HSPCs, we compared the ACD rates in MPP-, LMPP-, and GMP-enriched fractions of HC7-stained cells and analyzed MPP daughter cells at a single-cell level, both phenotypically and functionally.
Results
Discussion By using the HC7 anti-CD133 antibody, we demonstrate that almost all human HSPCs of the MPP fraction with confirmed lymphomyeloid and erythromyeloid developmental potential perform ACDs in vitro to create a lymphomyeloid CD34+CD133+CD45RA+ and a erythromyeloid CD34+CD133−CD45RA− daughter cell. In this context, we first comprehensively tested for the usability of the HC7 anti-CD133 antibody in human hematopoietic stem cell research. Side-by-side comparison of HC7 and the classical AC133 anti-CD133 antibody revealed that on fixed mitotic HSPCs, HC7, in contrast to AC133, allows detection of asymmetric CD133 distribution. Otherwise, no difference was detected regarding either their epitope expression on human HSPC subsets or their subcellular distribution on polarized or living mitotic HSPCs. Therefore, we conclude that the different appearance on fixed mitotic cells is an artifact caused by paraformaldehyde fixation rather than a difference in the HC7 and AC133 epitope distribution on HSPCs. Side-by-side comparison of HC7 or AC133 binding to human CD133+ HSPCs did not reveal a recognizable impact of any of these antibodies on the cell biological features of HSPCs, including their homing and repopulation capabilities in NOD/SCID mice. Thus, we consider the binding of both antibodies as functional neutral and conclude that they provide valuable tools for live-cell analyses of human CD133+ HSPCs.