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Steady-state blood CD34+ HSCs are CXCR4lowCD133+
still enhance the engraftment efficiency of SS-PB after ex vivo expansion, although this remains to be confirmed. On the other hand, recent studies have shown that CD34+ cells also home to the BM in an SDF1-CXCR4 axis-inde- pendent manner and that “priming factors”45 as well as “mild heat treatment” facilitate incorporation of CXCR4 into functional lipid rafts.46 This might constitute another strategy to enhance engraftment of SS-PB cells.
Concerning our observation of a close relationship between the expression of CXCR4 and CD9, CD9 has been implicated in the regulation of various physiological processes, including cell motility and adhesion. Trafficking and homing is a multistep process, as demonstrated for lymphocytes and myeloma cells, in which CD9 has been proven essential for transendothelial invasion.47 In human CB, CD9 is expressed by CD34+ cells and is regulated by SDF-1. Anti-CD9 antibody alters migratory and adhesive functions of CB CD34+ cells in vitro and CD9 neutraliza- tion impairs homing of transplanted CD34+ cells in NOD/SCID mice.8 The functional relationship between CD9 and CD26 on CDCXCR4bright cells remains to be elu- cidated.
In our hands, all the sorted SS-PB CD34+ subpopulations were CD90- and CD45RA-. This phenotype is associated with committed progenitor cells in BM and CB CD34+ cells since HSCs seem to be CD90+48 and/or CD45RA+.14 However, our CD34+CXCR4lowCD133+CD90-CD45RA- SS-PB cells are enriched in true HSCs, as proven by effi- cient secondary recipient hematopoietic engraftment. CD49f, claimed to be a specific marker of CB repopulating HSCs,15 is expressed on all CD34+CD133+ SS-PB cells whatever their CXCR4 expression.
Perhaps the most interesting information emerging from our study is the fact that all SS-PB HSCs exhibiting in vivo repopulating capacity (both ST- and LT-HSCs) are found to be exclusively a CD133+ population of CD34+ cells, highly concentrated in the CXCR4low population. This particular phenotypic determinant does not change after ex vivo expansion. With respect to the committed progenitors in the CD34+ population, our results (Figure 8) clearly show that before and after ex vivo expansion, CFU-GM and CFU- Mix reside exclusively in the CD133+ population, whereas BFU-E are present in both the CD133+ and CD133- popula- tions. This is in line with recent findings obtained with CB CD34+ cells.49-51 CD133 has long been considered a marker of stemness for CB, BM and M-PB cells although also expressed by most committed progenitor cells.16,18,49 Here, we show that CD133 could also be used for the enrich- ment of SS-PB HSCs. In CB, BM and M-PB cytokine-acti- vated CD34+ cells, CD133 is concentrated in the uropod of the polarized migrating cells.52 A functional relationship has been observed between CD133/prominin-1 and CXCR4 in specific membrane micro-domains of magnupo- dia,17 suggesting a favored cell migration towards the in vivo hematopoietic niche and, hence, engraftment. Since LT- HSCs are present only in the CD133+ fraction of CD34+CXCR4low SS-PB cells before and after expansion, the loss of this particular phenotypically-defined popula- tion in the course of ex-vivo manipulation could be indica- tive of a loss of the long-term repopulating capacity of the graft. Clinical scale CD133+ selection is also considered
among emerging strategies and alternative methods in clin- ical transplantation.53
BM mesenchymal stromal cell proliferation, but also fluctuation of the number of HSCs in peripheral blood are related to circadian oscillations.54 Since similar oscillations exist in humans,55 the circadian rhythm must be taken into consideration to optimize collection of SS-PB HSCs and HPCs.
Large, phenotypic and HPC analysis was performed on CD34+ cells isolated from SS-PB.56 Ex vivo culture of CD34+ SS-PB cells enhanced the total number of HSCs exhibiting in vivo repopulating capacity as well as their individual proliferative capacities, as shown by our limiting-dilution experiments. The maintenance of the lymphoid differen- tiation potential of repopulating HSCs after ex vivo culture is an additional important argument, since a shift towards predominant myeloid potential, as we detected in the rare CXCR4low and CD133- repopulating HSCs, has been found to occur during aging.57 In fact, our results suggest that reducing HSC differentiation capacity to the myeloid lin- eage represents a degree of HSC commitment. In this respect, aging is characterized by a higher proportion of more committed HSCs58 in a context of general “consump- tion” and is the first sign of imminent exhaustion of the system. This suggests that ex vivo expansion can provide an adequate tool to produce enough hematopoietic stem and progenitor cells to constitute a single hematopoietic graft from the contents of only one or two steady-state leukapheresis collections. The efficiency of the expansion procedure could, most likely, be further improved by using new approaches, for example the TAT-protein trans- duction peptide fused to regulatory factors or inhibition of HOXB4 degradation,59-62 which is the object of our ongo- ing work. Furthermore, the CD34neg fraction containing immuno-competent cells (T and B lymphocytes) can be preserved and an appropriate dose of these cells injected either during transplantation or later, depending on the need for an allogeneic immuno-effect. Furthermore, lym- phocyte efficiency can be enhanced and specified by ex vivo engineering.
Taken together, the results presented here might help in the design of novel, advanced graft generation, which could simultaneously provide efficient immuno- hematopoietic reconstitution and a graft-versus- tumor/leukemia effect. Future work in our laboratory aims to explore this strategy.
Acknowledgments
The authors thank to Mr Santiago Gonzalez, Mrs Valérie De Luca, Mrs Anaëlle Stum and Mr Vincent Pitard from Flow- Cytometry Platform SFR Transbiomed, Bordeaux University, France, for their precious help with cell sorting experiments. The help in first-line English editing of Mrs Elisabeth Doutreloux is also gratefully acknowledged. This manuscript was funded by an EFS (French Blood Institute - Etablissement Français du Sang) grant (n. 2016-01-IVANOVIC-AQLI) and French Biomedical Agency (Agence de la Biomédecine) grant (AOR “Greffe” 2015). IS would like to acknowledge support from the Liaison Committee between the Central Norway Health Authority (RHA) and the Norwegian University of Science and Technology (NTNU).
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