In line with our main finding that a single HSC may not be sufficient to generate detectable hematopoietic reconstitution in recipients, single-cell transplantations performed with various HSC-enriched populations may have chronically underestimated the total HSC frequencies. This study strongly suggests that C ≥1PMs should be routinely used to more accurately estimate HSC frequencies in LDTAs. 1a, 2a, and 2e ( P 1PMs fit better to the data than the SHPM. ![]() 5 In 7 of the 8 reanalyzed LDTAs, C ≥1PMs plausibly compete with the traditional SHPM, leading to significant changes in HSC frequency estimates compared with the SHPM. The validity of this new modeling approach is demonstrated by reassessing 8 previously published LDTAs 4, 5 aimed at comparing HSC frequencies in Hoxa-9 −/− versus wild-type mice (2 LDTAs) 4 and in Notch ligand–stimulated versus –unstimulated CD34 + cord blood cells (6 LDTAs). To address this problem, we demonstrate that it is possible to accurately quantitate HSCs, providing that the traditional SHPM is mathematically remodeled to turn to a new class of Poisson models termed multicell Poisson models (C ≥1PMs), which take into account the possibility of recipients misclassified as negative. This potential situation disqualifies the use of the SHPM. In this context, several recipients having received one, or even more than one, HSC(s) may be falsely classified as negative for reconstitution. Indeed, it is perfectly acceptable that the progeny of one HSC may be unable to reach this limit of detection imposed by standard flow cytometric analysis. 2 It turns out that the reliability of HSC frequency estimates is critically dependent on this hypothesis. 2 In particular, HSC frequency estimates traditionally rely on the SHPM, 1, 3 which posits that one donor HSC is sufficient to generate a progeny of detectable differentiated cells above a threshold value in hosts, usually considered approximately 1% of donor-derived cells as estimated by flow cytometry. Although limiting dilution transplantation assay (LDTA) in recipient animals coupled to the single-hit Poisson model (SHPM) is considered as one of the best methods for quantitating hematopoietic stem cells (HSCs), 1 investigators should be aware of the potential problems associated with this assay.
0 Comments
Leave a Reply. |