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Human platelet lysate (hPL) is considered a valid substitute to fetal bovine serum (FBS) in the expansion of mesenchymal stromal cells (MSC), and it is commonly produced starting from intermediate side products of whole blood donations. Through freeze–thaw cycles, hPL is highly enriched in chemokines, growth factors, and adhesion and immunologic molecules. Cell therapy protocols, using hPL instead of FBS for the expansion of cells, are approved by regulatory authorities without concerns, and its administration in patients is considered safe. However, published data are fairly difficult to compare, since the production of hPL is highly variable. This study proposes to optimize and standardize the hPL productive process by using instruments, technologies, and quality/safety standards required for blood bank activities and products. The quality and improved selection of the starting material (i.e., the whole blood), together with the improvement of the production process, guarantee a product characterized by higher content and quality of growth factors as well as a reduction in batch-to-batch variability. By increasing the number of freeze/thaw cycles from one (hPL1c) to four (hPL4c), we obtained a favorable effect on the release of growth factors from platelet α granules. Those changes have directly translated into biological effects leading to a decreasing doubling time (DT) of MSC expansion at 7 days (49.41 ± 2.62 vs. 40.61 ± 1.11 h, p < 0.001). Furthermore, mass spectrometry (MS)-based evaluation has shown that the proliferative effects of hPL4c are also combined with a lower batch-to-batch variability (10–15 vs. 21–31%) at the proteomic level. In conclusion, we have considered lot-to-lot hPL variability, and by the strict application of blood bank standards, we have obtained a standardized, reproducible, safe, cheap, and ready-to-use product.

Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity “endothelial GVHD” as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

BACKGROUND Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is burdened by life-threatening complications, with graft-versus-host disease (GvHD) being the major cause of morbility and mortality. Recently, clinical and physiopathologic evidences showed that vascular endothelium can be a target of GvHD in the early phase and circulating endothelial cells (CECs) represent surrogate markers of endothelial damage. METHODS Using the CellSearch System (Veridex LLC, Raritan, NJ), CECs were counted before (T1), after conditioning regimen (T2), at engraftment (T3), at GvHD onset (T4), and after steroid treatment (T5) in 40 patients (7 Hodgkin's Disease, 13 Acute Myeloblastic Leukemia, 5 Acute Lymphoblastic Leukemia, 8 Multiple Myeloma, 3 Chronic Lymphocytic Leukemia, 1 Non-Hodgkin Lymphoma, 1 Chronic Myeloid Leukemia, 2 Severe Aplastic Anemia) undergoing allo-HSCT. RESULTS The median CEC per milliliter at T1 was 20 (n=33, range 4-718), in comparison to a value of 2 (range, 1-14) in controls (P

The stromal progenitors of mesodermal cells, mesenchymal stromal cells (MSCs), are a heterogeneous population of plastic adherent fibroblast-like cells with extensive proliferative capacity and differentiation potential. Human MSCs have now been isolated from various tissues including bone marrow, muscle, skin, and adipose tissue, the latter being one of the most suitable cell sources for cell therapy, because of its easy accessibility, minimal morbidity, and abundance of cells. Bone marrow and subcutaneous or visceral adipose tissue samples were collected, digested with collagenase if needed, and seeded in Iscove's medium containing 5% human platelet lysate. Nonadherent cells were removed after 2-3 days and the medium was replaced twice a week. Confluent adherent cells were detached, expanded, and analyzed for several biological properties such as morphology, immunophenotype, growth rate, senescence, clonogenicity, differentiation capacity, immunosuppression, and secretion of angiogenic factors. The results show significant differences between lines derived from subcutaneous fat compared to those derived from visceral fat, such as the higher proliferation rate of the first and the strong induction of angiogenesis of the latter. We are convinced that the identification of the peculiarities of MSCs isolated from different tissues will lead to their more accurate use in cell therapy.

Summary Background X-linked severe combined immunodeficiency (SCIDX1) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common γ chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDX1 can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. Methods A male fetus was diagnosed as having SCIDX1 by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E resetting. Chimerism analysis was by HLA-DQα typing and γ-chain staining on cord blood. Findings A healthy 3·6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3·5 months of age the infant is well and his T-cell counts and function are normal. Interpretation In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSC) are multipotent cells that are considered one of the most promising product for cellular therapy in regenerative medicine. MSC have been obtained and expanded from bone marrow and umbilical cord blood in adequate amounts for clinical applications. Under the right conditions, MSC could migrate from bone marrow into the peripheral circulation; however MSC have not been routinely isolated from peripheral blood, and studies are rare and not conclusive. The aim of the present study was to evaluate mobilised peripheral blood (MPB), obtained from patients undergoing apheresis collection of circulating hematopoietic progenitor cells, as a potential source of MSC for clinical applications. MPB samples (500–900 × 106 cells, N = 17) were separated by negative lineage-depletion immunoselection (RosetteSep). Selected cells were seeded in multi-well plates at low density in MesenCult Basal Medium without and with different combinations of growth factors (EGF, PDGF-BB, b-FGF). On reaching confluence, adherent cells were detached by 0.25% trypsin-EDTA treatment and replated for at least two passages. At each passage, surface antigen expression was analyzed by flowcytometry (CD45, CD34, CD105, CD44, CD73, CD166, CD31, HLA-DR and VE-caderine). Following immunoselection 9.5–17.1 × 106 cells were recovered from MPB samples. Cultured cells reached confluency in 3–4 weeks on first passage and in two weeks thereafter. Immunophenotyping showed negativity for CD45 antigen. The absence of growth factors in culture medium conditioned MSC growth capability, while the addition of PDGF-BB+EGF or b-FGF was able to boost the number of CD45−/CD73+/CD90+ cells in culture (see figure). However expansion remains still sub-optimal, having been reached in 8/17 samples. In conclusion, we demonstrate that MSC can be obtained from MPB, but expansion requires longer time period and appears more difficult compared to bone marrow. Therefore, further studies need to be conducted to find better culture conditions and optimal growth factor combinations to support MPB-derived MSC expansion. Download : Download high-res image (83KB) Download : Download full-size image Figure

X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis.A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood.A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal.In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.