Peripheral blood stem cell mobilization with pegylated granulocyte colony stimulating factor in children.

BACKGROUND AND AIM: We report here our experience of using pegylated granulocyte colony stimulating factor (peg-GCSF) for peripheral blood stem cell (PBSC) mobilization in children. METHODS AND RESULTS: A total of nine children suffering from high-risk/relapsed solid tumors were mobilized with chemotherapy and peg-GCSF (100 microgram/kg single dose). Mean age was 7.7 years (range 2-15 years).The mean time from peg-GCSF administration to PBSC harvest was 9.7 days. Adequate stem cells (median dose 26.9 million/kg) could be harvested in all children by a single apheresis procedure. No major adverse events observed. CONCLUSION: It is feasible and safe to mobilize PBSC with peg-GCSF in children with cancer.

Influence of laboratory procedures on postthawing cell viability and hematopoietic engraftment after autologous peripheral blood stem cell transplantation.

BACKGROUND: The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS: This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS: The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION: The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.

CD34+ cell dose effects on clinical outcomes after T-cell replete haploidentical allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia using peripheral blood stem cells. A study from the acute leukemia working Party of the European Society for blood and marrow transplantation (EBMT).

Previous observations have reported controversial conclusions regarding cell dose and survival endpoints after allogeneic hematopoietic stem cell transplantation (HSCT). We conducted a retrospective analysis on 414 adult patients (median age 54 years, range, 18-74 years) with acute myeloid leukemia (AML) in first and second complete remission. They received a T-cell replete allogeneic HSCT from haploidentical donors, using peripheral blood stem cells, between 2006-2018. Median number of infused CD34+ was 6.58 × 106 /kg (range, 2.2-31.2 × 106 /kg). Graft-vs-host disease (GVHD) prophylaxis was post-transplant cyclophosphamide in 293 patients and anti-lymphocyte serum in 121 patients. Conditioning was myeloablative in 179 patients and reduced-intensity in 235 patients. After a median follow-up of 23.3 months (range, 12.1-41.8 months), 2-year overall survival (OS) was 64.5% (95% CI 59.3%-69.7%) with leukemia-free survival (LFS) of 57.3% (95% CI 51.8%-62.7%) and non-relapse mortality (NRM) of 23.3% (95% CI 19%-27.7%). Grades III-IV acute GVHD day+100 incidence was 14.6% while extensive chronic GVHD was 14.4% at 2-years. Thirteen (3.2%) patients experienced graft failure. We found the optimal CD34+/kg threshold defining high (n = 334) vs low cell dose (n = 80) at 4.96 × 106 . Recipients of >4.96 × 106 /kg CD34+ cells experienced less NRM (Hazard ratio [HR] 0.48; 95% CI 0.30-0.76) and prolonged LFS (HR 0.63; 95% CI 0.43-0.91) and OS (HR 0.60; 95% CI 0.40-0.88) compared to those in the lower cell dose cohort. Larger cohort studies are needed to confirm these findings.

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes.

INTRODUCTION: Cell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34+ quantification; (ii) percentage of CD34+ viability and (iii) evaluation of HSC functional ability to form colony forming unit-granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34+, using a Fluorescent Labeled Inhibitor of CAspases (FLICA). METHODS: Caspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34+, CD3+, CD14+ and CD56+ cells. Viable 7AAD-/FLICA+ cells were then correlated with various parameters. RESULTS: We showed a significant caspase pathway activation, with 23% CD34+/7AAD-/FLICA+ cells after thawing, compared with the 2% described in fresh CD34+ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3+, CD56+ and CD14+ cells. We also report a significant correlation between the rate of CD34+/7AAD-/FLICA+ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). DISCUSSION: Our results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3+ and CD56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).

Simultaneous in vitro generation of human CD34+-derived dendritic cells and mast cells from non-mobilized peripheral blood mononuclear cells.

Dendritic cells (DCs) and mast cells (MCs) are key players of the immune system, often coming in close proximity in peripheral tissues. The interplay of these cells is, however, still poorly understood, especially with regards to human cells. The reason for that is the absence of a well established in vitro human cell-based study system that would allow a simultaneous preparation of both cell types. In this study, we show a method for simultaneous generation of DCs and MCs from CD34+ stem cell progenitors that were isolated from the non-adherent fraction of non-mobilized peripheral blood mononuclear cells of healthy donors. We observed that combining stem cells factor (SCF), IL-3 and GM-CSF in serum-free StemPro-34 medium allowed CD34+ cells isolated from an equivalent of 450 ml of peripheral blood to expand to 10-92 × 106 cells after 7 weeks of culturing. These cultures comprised of 6-53% of DCs and 1-21% of MCs as determined by the expression of, respectively, CD11c/HLA-DR or CD117/FcεRI. The DCs were CD1a-CD14-, did not express co-stimulatory molecules CD80 and CD83 and chemokine receptor CCR7. However, the DCs expressed co-stimulatory molecule CD86, and had a capacity to uptake dextran, phagocyte latex particles and induce alloreactivity. MCs, on the other hand, degranulated after crosslinking of FcεRI-bound IgE as determined by the externalization of CD107b. Collectively, our data show that CD34+-derived human DCs and MCs can be generated in a single culture using CD34+ cells isolated from non-mobilized human peripheral blood and that this method may allow ex vivo studies on DC-MC interplay in human system.

Blood Stem Cell Activity Is Arrested by Th1-Mediated Injury Preventing Engraftment following Nonmyeloablative Conditioning.

T cells are widely used to promote engraftment of hematopoietic stem cells (HSCs) during an allogeneic hematopoietic cell transplantation. Their role in overcoming barriers to HSC engraftment is thought to be particularly critical when patients receive reduced doses of preparative chemotherapy and/or radiation compared with standard transplantations. In this study, we sought to delineate the effects CD4+ cells on engraftment and blood formation in a model that simulates clinical hematopoietic cell transplantation by transplanting MHC-matched, minor histocompatibility-mismatched grafts composed of purified HSCs, HSCs plus bulk T cells, or HSCs plus T cell subsets into mice conditioned with low-dose irradiation. Grafts containing conventional CD4+ T cells caused marrow inflammation and inhibited HSC engraftment and blood formation. Posttransplantation, the marrows of HSCs plus CD4+ cell recipients contained IL-12-secreting CD11c+ cells and IFN-γ-expressing donor Th1 cells. In this setting, host HSCs arrested at the short-term stem cell stage and remained in the marrow in a quiescent cell cycling state (G0). As a consequence, donor HSCs failed to engraft and hematopoiesis was suppressed. Our data show that Th1 cells included in a hematopoietic allograft can negatively impact HSC activity, blood reconstitution, and engraftment of donor HSCs. This potential negative effect of donor T cells is not considered in clinical transplantation in which bulk T cells are transplanted. Our findings shed new light on the effects of CD4+ T cells on HSC biology and are applicable to other pathogenic states in which immune activation in the bone marrow occurs such as aplastic anemia and certain infectious conditions.

Cryopreservation in Closed Bag Systems as an Alternative to Clean Rooms for Preparations of Peripheral Blood Stem Cells.

Autologous and allogeneic stem cell transplantation (SCT) represents a therapeutic option widely used for hematopoietic malignancies. One important milestone in the development of this treatment strategy was the development of effective cryopreservation technologies resulting in a high quality with respect to cell viability as well as lack of contamination of the graft.Stem cell preparations have been initially performed within standard laboratories as it is routinely still the case in many countries. With the emergence of cleanrooms, manufacturing of stem cell preparations within these facilities has become a new standard mandatory in Europe. However, due to high costs and laborious procedures, novel developments recently emerged using closed bag systems as reliable alternatives to conventional cleanrooms. Several hurdles needed to be overcome including the addition of the cryoprotectant dimethylsulfoxide (DMSO) as a relevant manipulation. As a result of the development, closed bag systems proved to be comparable in terms of product quality and patient outcome to cleanroom products. They also comply with the strict regulations of good manufacturing practice.With closed systems being available, costs and efforts of a cleanroom facility may be substantially reduced in the future. The process can be easily extended for other cell preparations requiring minor modifications as donor lymphocyte preparations. Moreover, novel developments may provide solutions for the production of advanced-therapy medicinal products in closed systems.

Blood stem cells: from beginning to end.

In June 2016, around 200 scientists from all over the world gathered at EMBL headquarters in Heidelberg, Germany to discuss the recent advances in hematopoietic stem cells from three different angles: developmental, adulthood and aging. The meeting, aptly named 'Hematopoietic stem cells: from the embryo to the aging organism' also covered cutting-edge technologies applied to this subject, such as single-cell analysis, reprogramming and imaging. This Meeting review summarizes the exciting work that was presented and covers the main themes that emerged from the meeting.

The effect of bisphosphonate treatment on osteoclast precursor cells in postmenopausal osteoporosis: The TRIO study.

Bisphosphonates are used to treat bone disease characterised by increased bone resorption by inhibiting the activity of mature osteoclasts, resulting in decreased bone turnover. Bisphosphonates may also reduce the population of osteoclast precursor cells. Our aims were to investigate the effect of bisphosphonates on i) osteoclast precursor cells and ii) circulating cytokine and cytokine receptor in postmenopausal women with osteoporosis compared with healthy premenopausal women. Participants were 62 postmenopausal women (mean age 66) from a 48-week parallel group trial of bisphosphonates. They received ibandronate 150mg/month (n=22), alendronate 70mg/week (n=19) or risedronate 35mg/week (n=21). Fasting blood was collected at baseline, weeks 1 and 48. At baseline, blood was also collected from 25 healthy premenopausal women (mean age 37) to constitute a control group. Peripheral blood mononuclear cells were extracted and stained for CD14, M-CSFR, CD11b and TNFRII receptors. Flow cytometry was used to identify cells expressing CD14+ and M-CSFR+ or CD11b+ or TNFRII+. RANKL and OPG were measured to evaluate potential mediation of the bisphosphonate effect. After 48weeks of treatment, there was a decrease in the percentage of cells expressing M-CSFR and CD11b receptors by 53% and 49% respectively (p<0.01). Cells expressing M-CSFR and CD11b were decreased with ibandronate and risedronate after 48weeks to the lower part of the premenopausal reference interval. These effects were not significantly different between each of the treatment groups. There was no significant effect on RANKL and OPG throughout the study period. Bisphosphonates inhibit bone resorption in the short-term by direct action on mature osteoclasts. There is also a later effect mediated in part by a reduction in the population of circulating osteoclast precursors.