Search

Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects in vitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1's binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin-CD34- and Lin-CD34+ cells were isolated from granulocyte colony-stimulating factor-primed peripheral blood (PB) cells of healthy donors. The CD34- cell fraction did not contain either clonogenic cells in semisolid culture or long-term culture initiating cells (LTC-IC). However, stroma-dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC-IC. Significantly higher percentages of quiescent G0 cells and lower percentages of cycling G1 cells were found in Lin-CD34- cells when compared with Lin-CD34+ cells. Kinetic quiescence of Lin-CD34- cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27Kip1 and p21(cip1/waf1). Cytokine-mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long-term engraftment in immunocompromised non-obese diabetic (NOD)/recombination activating gene 1null and NOD/severe combined immunodeficient-beta2microglobulin(null) mice injected with CD34+ cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell-cycle recruitment and downregulation of p27Kip1 in vitro and higher engraftment capacity in vivo.

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.

Umbilical cord blood (UCB) transplant represents a promising therapeutic approach, nevertheless this procedure has been so far almost exclusively used in pediatric patients because of the reduced volume of UCB units. The availability of larger numbers of early and late hematopoietic progenitors by ex vivo amplification procedure may allow the use of UCB in adults and improve the rate and time to engraftment. We describe a stroma-free liquid culture system that induces a 10-fold increase of CD34+ cells and hematopoietic progenitors after 8 days in vitro amplification. The presence of flt3L is essential to preserve and amplify the early stem cell compartment identified by the phenotype CD34+Thy-1+CD45RO+.

In this study, we assessed the functional and kinetic characteristics of highly purified hematopoietic CD34+ cells from the apheresis products of 16 normal donors undergoing glycosylated granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation in allogeneic recipients. Mobilized CD34+ cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) CSF in vitro and the content of very primitive long-term culture initiating cells (LTC-IC). In addition, the cycling status of circulating CD34+ cells, including committed clonogenic progenitor cells and the more immature LTC-IC, was determined by the cytosine arabinoside (Ara-C) suicide test and the acridine orange flow cytometric technique. By comparison, bone marrow (BM) CD34+ cells from the same individuals were studied under steady-state conditions and during G-CSF administration. Clonogenic assays in methylcellulose showed the same frequency of colony-forming unit cells (CFU-C) when PB-primed CD34+ cells and BM cells were stimulated with phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM). However, mobilized CD34+ cells were significantly more responsive than their steady-state BM counterparts to interleukin-3 (IL-3) and stem cell factor (SCF) combined with G-CSF or IL-3 in presence of erythropoietin (Epo). In cultures added with SCF, IL-3, and Epo, we found a mean increase of 1.5- +/- 1-fold (standard error of the mean [SEM]) of PB CFU-granulocyte-macrophage and erythroid progenitors (burst-forming units-erythroid) as compared with BM CD34+ cells (P < .05). Conversely, circulating and BM megakaryocyte precursors (CFU-megakaryocyte) showed the same clonogenic efficiency in response to IL-3, granulocyte-macrophage-CSF and IL-3, IL-6, and Epo. After 5 weeks of liquid culture supported by the engineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we reported 48.2 +/- 35 (SEM) and 62.5 +/- 54 (SEM) LTC-IC per 10(4) CD34+ cells in PB and steady-state BM, respectively (P = not significant). The Ara-C suicide assay showed that 4% +/- 5% (standard deviation [SD]) of committed precursors and 1% +/- 3% (SEM) of LTC-IC in PB are in S-phase as compared with 25.5% +/- 12% (SD) and 21% +/- 8% (SEM) of baseline BM, respectively (P < .001). However, longer incubation with Ara-C (16 to 18 hours), in the presence of SCF, IL-3 and G-CSF, or IL-6, showed that more than 60% of LTC-IC are actually cycling, with no difference being found with BM cells. Furthermore, studies of cell-cycle distribution on PB and BM CD34+ cells confirmed the low number of circulating progenitor cells in S- and G2M-phase, whereas simultaneous DNA/RNA analysis showed that the majority of PB CD34+ cells are not quiescent (ie, in G0-phase), being in G1-phase with a significant difference with baseline and G-CSF-treated BM (80% +/- 5% [SEM] v 61.9% +/- 6% [SEM] and 48% +/- 4% [SEM], respectively; P < .05). Moreover, G-CSF administration prevented apoptosis in a small but significant proportion of mobilized CD34+ cells. Thus, our results indicate that mobilized and BM CD34+ cells can be considered equivalent for the frequency of both committed and more immature hematopoietic progenitor cells, although they show different kinetic and functional profiles. In contrast with previous reports, we found that PB CD34+ cells, including very primitive LTC-IC, are cycling and ready to progress into S-phase under CSF stimulation. This finding should be taken into account for a better understanding of PBSC transplantation.

Management of acute lymphoblastic leukemia (ALL) patients may include growth factors (GFs) to reduce post-chemotherapy aplasia. A potential risk of GF administration is a stimulatory signal on the leukemic population. In the present study we investigated the proliferative and programmed cell death (PCD) effect of two cytokines that have recently entered clinical use, stem cell factor (SCF) and the granulocyte colony stimulating factor/IL-3 fusion molecule (PIXY-321), on 14 ALL samples. The activity of IL-7, a cytokine involved in the regulation of ALL cell proliferation, was also tested alone and in combination with these two cytokines. Using the acridine orange flow cytometric technique and the clonogenic assay, we showed that none of these cytokines was capable of significantly increasing the mean percentage of S-phase cells and CFU-L number. A mean decrease of G0 cells from 60.6% to 52.6% (p = 0.02), coupled by a significant increase of G1 cells from 28.2% to 37.9% (p = 0.003) was demonstrated in the presence of PIXY-321. IL-7 alone and in combination with either PIXY-321 or SCF induced similar changes in the percentage of cells in G0 and G1. SCF showed no activity on G0 depletion. When each individual samples was analyzed separately, some heterogeneity was observed. An increase of S phase was recorded in a proportion of cases after SCF and PIXY-321 exposure. However, none of the cytokines evaluated by a clonogenic assay following liquid culture was capable of maintaining or promoting self-renewal of leukemic precursors, as determined by plating fresh cells at time 0. Detection of cytokine effects of apoptosis showed that SCF and PIXY-321 did not significantly reduce the mean percentage of cells in PCD, whereas a significant protective effect was observed in the presence of IL-7 (p = 0.02). We conclude that PIXY-321 and, to a further extent, SCF fail to induce leukemic lymphoid cell proliferation, and do not protect cells from entering apoptosis. These in vitro findings may be useful for ALL clinical trial design.