We recently developed a clinical grade ex vivo cord blood (CB) expansion procedure enabling a massive amplification of hematopoietic progenitors without any loss of stem cell potential as revealed on the basis of serial engraftment of Nod/SCID mice (Ivanovic et al, Cell Transplant. 2011). This procedure, in line with our concept "Oxygen Stem Cell Paradigm" (Ivanovic, J Cell Physiol 2009), is based on Day 14 liquid cultures of CB CD34+ cells, in medium Macopharma HP01 (containing several antioxidant molecules and nicotinamide) and in presence of Stem Cell Factor - SCF (100 ng/ml), fms-Related Tyrosine Kinase 3 – Ligand - FLT3-ligand (100ng/ml), Megakaryocyte Growth and Developmental Factor - MGDF (100 Ng/ml) (these two cytokines acting in favor of HIF-1alpha transcript stabilization) and Granulocyte – Colony Stimulating Factor -G-CSF (10 Ng/ml). This cocktail had to be modified due to the commercially unavailability of clinical grade MGDF molecule. So, MGDF was replaced by Thrombopoietin - TPO in five-fold lower dose (20 ng/ml) and culture time was reduced to 12 days (Duchez et al Cell Transplant 2012). That way, a mean expansion fold of 400, 80, and 150 was obtained for total cells, CD34+ cells and Colony Forming Cells – CFC, respectively. This amplification was associated with a slight enhancing effect on stem cells (Scid Repopulating Cells - SRC). These are the ultimate pre-clinical modifications of a clinical grade expansion protocol which is already employed in an ongoing clinical trial (adult allogeneic context) started in 2010). The preliminary results are encouraging – rapid and durable hematopoietic reconstitution after injection of one ex vivo expanded CB unit only.

Twenty four patients (pts) with planned autologous stem cell transplantation for lymphoma diseases (Hodgkin's disease=4; non-Hodgkin's lymphoma=20) received chemotherapy (CT) (Induction CT=3 and salvage regimen= 21) followed by a fixed single dose (6 mg) administration of Pegfilgrastim (PF) after the last day of CT for peripheral blood stem cell collection (PBSC) (target cell dose of 3 2×106 CD34+/kg). Median age was 53 yrs (24–68) and median weight was 72, 5 kg (45–98). Among the 24 pts, 7 received more than 2 lines of CT regimens. The injection of PF was well tolerated. Median time interval between day 1(D1) of the cycle of CT mobilization and first leukapheresis session was 14 days (10–18) while the median time interval between injection of PF and first leukapheresis session was 9 days (6–13). Stem cell collection was started when the absolute number of circulating CD34+ cells was >10×106/L and performed with standard volume leukapheresis. Median CD34+ cells level at D1 of leukapheresis was 35, 5/mm3 (11–320) and interestingly, more than 35 % of pts could reach this median level of CD34+ early after PF injection (around D6). Notably, 22 pts reached the target cell dose in 2 sessions of leukapheresis or less (10 pts after 1 session, 10 other pts after 2 sessions, 2 pts after 3 and 4 sessions respectively). The median number of leukapheresis sessions was 2(1–4) and the median CD34+ cells harvested was 4×106/kg (0,8–26,6). Two pts (DLBCL = 1 and FL = 1) could not reach the level of CD34+ required to start leukapheresis and both became secondary refractory to CT. In univariate analysis, PBSC collection of > 4×106/kg was highly correlated with pts who started their collection at D9 of PF administration (P=0,01) and with those presenting a CD34+ cells level > 35.5/mm3 at D1 of leukapheresis (P=0,033). White blood cells level higher than 9 G/l was also predictive of circulating CD34+ cells >35,5/mm3 (P=0,033). These data suggest that PF may represent an attractive option for PBSC mobilization particularly for pts with lymphoma when optimal compliance of frequent sequential regimens of CT is required. We also emphasize that stem cell mobilization is effective even in pts in second or subsequent salvage CT regimen. Importantly, the circulating CD34+ count should be performed from D6 of PF administration. The presentation will include the updated data.

Cord blood (CB) units are increasingly used for allogeneic transplantation. Cell dose, a major factor for CB selection, is evaluated before freezing by each CB bank, using various techniques. This may introduce variability and affect the prediction of cell recovery after thawing, or haematopoietic reconstitution. Forty-two children were transplanted at the same institution with unrelated CB units. All units were thawed and evaluated at the same cell therapy facility, using standard procedures. We investigated: (i) factors that affect cell loss after thawing, and (ii) the importance of CD34(+) cell doses. Prefreeze and post-thaw CD34(+) cell doses were statistically correlated, thus suggesting that variability in numeration techniques used by different CB banks does not compromise the biological and clinical value of these figures. CD34(+) cell recovery appeared to be correlated with the absolute number of CD34(+) cells per frozen bag. Infused CD34(+) is the cell dose that better correlates with platelet reconstitution delay; in addition, when using a quartile comparison, haematopoietic recovery appeared to be related with prefreeze and post-thaw CD34(+) cell doses. We conclude that enumeration of CD34(+) cells in CB units is of biological significance, and may help select CB units and identify patients at risk of delayed recovery.

BACKGROUND: Used leukodepletion filters (LDFs), containing billions of white blood cells (WBCs), are discarded. Because the steady-state blood contains low quantities of stem and progenitor cells that are retained in LDFs, the viability and the functional properties of mononuclear cells (MNCs) and CD34+ cells recovered from LDFs were investigated.STUDY DESIGN AND METHODS: WBCs were recovered from LDFs by use of a closed system. MNCs and CD34+ cells were isolated from freshly LDF-recovered WBCs or after their overnight incubation. The CD34+ cells were enumerated, as well as the number of colony-forming unit (CFU)-granulocyte-macrophage, burst-forming unit-erythroid, and CFU-Mixed. The expansion in clinical-scale volume cultures (serum-free medium plus stem cell factor, granulocyte-colony-stimulating factor, and megakaryocyte growth and development factor) was performed starting from MNCs, freshly isolated CD34+ cells, and CD34+ cells isolated after overnight incubation of WBCs. The erythroid, megakaryocytic, eosinophilic, and monocyte-myelocytic lineage differentiation of LDF-recovered CD34+ cells was challenged in liquid cultures by adding relevant cytokines.RESULTS: Nearly 450 x 10(3) viable CD34+ cells were recovered per LDF. These cells exhibit unimpaired colony-forming ability. It is possible to expand these cells ex vivo, but their response to cytokines is different compared to mobilized peripheral blood and cord blood CD34+ cells. Thus, further work is necessary to optimize their ex vivo expansion. These cells give rise to the mature cells and precursors of erythroid, megakaryocytic, eosinophilic, and monomyelocytic lineage in liquid cultures.CONCLUSION: MNCs and CD34+ cells recovered from the LDFs exhibit unimpaired functional capacities. Recent development of ex vivo technologies for expansion, retrodifferentiation, and differentiation reinforces the value in cell therapy of these LDG-recovered peripheral blood progenitor cells that are routinely discarded.

BACKGROUND:The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated. STUDY DESIGN AND METHODS:Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity. RESULTS:A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed. CONCLUSION:Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.

In the present work, we tested the hypothesis that liquid cultures (LCs) of cord blood CD34(+) cells at an appropriate low O-2 concentration could simultaneously allow colony-forming cell (CFC) expansion and nonobese diabetic/severe combined immunodeficiency mice-repopulating cell (SRC) maintenance. We first found that 3% was the minimal O-2 concentration, still allowing the same rate of CFC expansion as at 20% O-2. We report here that 7-day LCs of cord blood CD34(+) cells at 3% O-2 maintain SRC better than at 20% O-2 and allow a similar amplification of CFCs (35- to 50-fold) without modifying the CD34(+) cell proliferation. Their phenotypic profile (antigens: HLA-DR, CD117, CD33, CD13, CD11b, CD14, CD15, and CD38) was not modified, with exception of CD133, whose expression was lower at 3% O-2. These results suggest that low O-2 concentrations similar to those found in bone marrow participates in the regulation of hematopoiesis by favoring stem cell-renewing divisions. This expansion method that avoids stem cell exhaustion could be of paramount interest in hematopoietic transplantation by allowing the use of small-size grafts in adults.

BACKGROUND: Mobilized PBPCs, detectable on the basis of CD34 expression, can be collected on various cell separators. The CD34+ cell collection efficiencies of two cell separators (CS‐3000+ and Amicus, Baxter) were tested on two comparable groups of oncology patients. STUDY DESIGN AND METHODS: Leukapheresis assisted by the standard manufacturer's software and variables settings was performed in 37 (CS‐3000+) and 34 (Amicus) patients (total of 83 and 67 collections, respectively) after chemotherapy plus G‐CSF treatment. RESULTS: The total CD34+ cell count per leukapheresis components as well as per kg of patient's body weight were twofold higher by using the Amicus than the CS‐3000+ device. Platelet contamination in Amicus components was twice as low compared to the CS3000+. Mean Amicus CD34+ collection efficiency (CD34+eff) (54.9 ± 27.2%) was significantly higher (p < 0.015) than the CS‐3000+ (46.4 ± 16.7%) one. However, Amicus CD34+eff decreased progressively as the peripheral blood CD34+ concentrations increases over 200 CD34+ cells per µL. A parallel increase in the WBC counts in these cases seems to be the principal cause of decrease in CD34+eff (evident for WBCs >40 × 10 3 /µL and most pronounced for WBCs >60 × 10 3 /µL). CONCLUSIONS: Mean CD34+eff and CD34+ cell yields were better on Amicus than on CS‐3000+. CD34+eff of Amicus, however, seems to be related to the initial WBC counts, decreasing progressively when WBC increased over 4 × 10 3 per µL that coincided with the increase in CD34+ cell concentrations. For these cases, the volume and duration of cycles should be adapted to optimize CD34+ collections by using Amicus separators.