Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrowederived mesenchymal stromal cells

Abstract Background aims. A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Methods. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. Results. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. Conclusions. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS

Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

Dexamethasone scheduling in MNC culture. The supplement was added in standard medium until the detection of MSC colonies (n = 16 CB units) or alternatively added for the first week of MNC culture only (n = 34). (DOCX 120 kb

Prenatal detection of trisomy 8 mosaicism: Pregnancy outcome and follow up of a series of 17 consecutive cases

OBJECTIVE: To study the outcome of a series of individuals with prenatal detection of trisomy 8 mosaicism by chorionic villus sampling (CVS) and/or amniocentesis. STUDY DESIGN: The databases of two Italian genetics units were reviewed to identify all consultations requested during pregnancy because of trisomy 8 mosaicism. To evaluate the pregnancy outcome, the regional registry of congenital malformations (including terminations of pregnancies) was consulted; additional follow-up data were collected by a telephone interview. The following outcomes were analysed: delivery, pre- and post-natal growth, psychomotor development, major malformations, other diseases/complications. RESULTS: A total of 17 consecutive cases of trisomy 8 mosaicism were identified. Fourteen cases were first detected among women undergoing prenatal diagnosis by CVS; the remaining ones were identified among women who underwent amniocentesis. In most cases diagnosed by CVS, the chromosomal anomaly was only detected in long-term cell cultures (10/14) and was not confirmed by amniocentesis (11/13). There were two terminations of pregnancy and 15 live births; no major birth defects were observed among live born infants and only a case with prenatal and postnatal growth retardation was observed (mean age at follow-up interview was 5.9 years). CONCLUSION: Our data showed an overall positive prognosis for cases with an apparent confined placental mosaicism and those with low-level mosaicism in amniotic fluid if no congenital anomalies were detected by foetal ultrasound examinations. However, larger studies are warranted to better define the associated risk of neurodevelopmental anomalies

Genetics and Autosomal Dominant Polycystic Kidney Disease Progression

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, accounting for 10% of European patients on renal replacement therapy. In the previous years, many approaches to slow the progression of ADPKD were studied and many clinical trials published. In addition to having diagnostic role, the description of the genotype is even important to predict the progression of the disease and contributes, combined with several other factors, to a more precise patients classification. With the availability of disease-modifying drugs, "fast progression factors" are needed to early recognize those patients who would likely progress, before cyst growth reaches a critical value. ERA-EDTA working group on inherited kidney disorders included a series of recommendations resulting in a hierarchical decision algorithm to select patients who are most likely to benefit from the treatment. Beyond diagnosis, we will also discuss the important role of genetics in ADPKD progression and management

Evans syndrome secondary to chronic lymphocytic leukaemia: presentation, treatment, and outcome

Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome.Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome

CVVHD treatment with CARPEDIEM: small solute clearance at different blood and dialysate flows with three different surface area filter configurations

The CARdiorenal PEDIatric EMergency (CARPEDIEM) machine was originally designed to perform only continuous venovenous hemofiltration (CVVH) in neonatal and pediatric patients. In some cases, adequate convective clearance may not be reached because of a limited blood flow. In such conditions, the application of diffusive clearance [continuous venovenous hemodialysis (CVVHD)] would help optimize blood purification. In this study, the CARPEDIEM\u2122 machine was modified to enable the circulation of dialysis through the filter allowing testing of the performance of CARPEDIEM\u2122 machine in CVVHD

Additional file 10: Figure S7. of Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

SL-CBMSC immunophenotypic analysis. Characterization of SL-MSC (n = 6) by flow cytometry using a panel of 14 cell surface markers. Boxes extend from 25th percentile to the 75th percentile, the middle line represents median value and the whiskers extend from minimum to maximum values. Data are displayed as rMFI on the unstained control. (DOCX 104 kb

Additional file 5: Table S2. of Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

List of antibodies used for immunophenotypic analysis. (DOCX 14 kb