The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing–thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.
Summary. Background: Presence of bleeding symptoms, inheritance and reduced von Willebrand factor (VWF) contribute to the diagnosis of type 1 von Willebrand disease (VWD). However, quantitative analysis of the importance of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) levels in the diagnosis is lacking. Objectives: To evaluate the relative contribution of VWF measurement to the diagnosis of VWD. Patients and methods: From the MCMDM-1VWD study cohort, 204 subjects (considered as affected by VWD based on the enrolling Center diagnoses and the presence of linkage with the VWF locus) were compared with 1155 normal individuals. Sensitivity, specificity and diagnostic positive likelihood ratios (LR) of VWF:Ag and VWF:RCo were computed. Results: ABO blood group was the variable most influencing VWF levels, but adjustment of the lower reference limit for the ABO group did not improve sensitivity and specificity of VWF:Ag or VWF:RCo. The lower reference limit (2.5th percentile) was 47 IU dL ) of VWF only marginally indicated the probability of VWD. Conclusions: Although the conventional 2.5 lower percentile has good sensitivity and specificity, only VWF:Ag or VWF:RCo values below 40 IU dL )1 appear to significantly indicate the likelihood of type 1 VWD. The LR profile of VWF level could be used in a diagnostic algorithm.
BackgroundThe use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted. Platelet derivatives are valid FBS substitutes due to their content of growth factors that can be released disrupting the platelets by physical methods or physiological stimuli. We compared platelet derivatives produced by freezing/thawing (platelet lysates, PL) or after CaCl2 activation (platelet releasate surnatant rich in growth factors, PR-SRGF) for their content in growth factors and their ability to support the ex vivo expansion of BM-MSC.MethodsThe cytokine content in the two platelet derivatives was evaluated. BM-MSC were expanded in complete medium containing 10, 7.5 and 5% PL or PR-SRGF and the cell phenotype, clonogenic capacity, immunomodulation properties and tri-lineage differentiation potential of the expanded cells in both media were investigated.ResultsThe concentration of PDGF-AB, PDGF-AA, PDGF-BB in PR-SRGF resulted to be respectively 5.7×, 1.7× and 2.3× higher compared to PL. PR-SRGF promoted a higher BM-MSC proliferation rate compared to PL not altering BM-MSC phenotype. Colony forming efficiency of BM-MSC expanded in PR-SRGF showed a frequency of colonies significantly higher than cells expanded in PL. BM-MSC expanded in PL or PR-SRGF maintained their immunomodulatory properties against activated lymphocytes even if BM-MSC expanded in FBS performed significantly better.ConclusionsThe method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance. The standardization of the production process of platelet derivatives and the definition of their release criteria requires further investigation.
BackgroundMesenchymal stromal cells (MSC) are a heterogeneous population of multipotent progenitors used in the clinic because of their immunomodulatory properties and their ability to differentiate into multiple mesodermal lineages. Although bone marrow (BM) remains the most common MSC source, cord blood (CB) can be collected noninvasively and without major ethical concerns. Comparative studies comprehensively characterizing the MSC phenotype across several tissue sources are still lacking. This study provides a 246-antigen immunophenotypic analysis of BM- and CB-derived MSC aimed at identifying common and strongly expressed MSC markers as well as the existence of discriminating markers between the two sources.MethodsBM-MSC (n = 4) were expanded and analyzed as bulk (n = 6) or single clones isolated from the bulk culture (n = 3). CB-MSC (n = 6) were isolated and expanded as single clones in 5/6 samples. The BM-MSC and CB-MSC phenotype was investigated by flow cytometry using a panel of 246 monoclonal antibodies. To define the markers common to both sources, those showing the smallest variation between samples (coefficient of variation of log2 fold increase ≤ 0.5, n = 59) were selected for unsupervised hierarchical cluster analysis (HCL). Differentially expressed markers were identified by directly comparing the expression of all 246 antigens between BM-MSC and CB-MSC.ResultsBased on HCL, 18 markers clustered as strongly expressed in BM-MSC and CB-MSC, including alpha-smooth muscle antigen (SMA), beta-2-microglobulin, CD105, CD13, CD140b, CD147, CD151, CD276, CD29, CD44, CD47, CD59, CD73, CD81, CD90, CD98, HLA-ABC, and vimentin. All except CD140b and alpha-SMA were suitable for the specific identification of ex-vivo expanded MSC. Notably, only angiotensin-converting enzyme (CD143) was exclusively expressed on BM-MSC. CD143 expression was tested on 10 additional BM-MSC and CB-MSC and on 10 umbilical cord- and adipose tissue-derived MSC samples, confirming that its expression is restricted to adult sources.ConclusionsThis is the first study that has comprehensively compared the phenotype of BM-MSC and CB-MSC. We have identified markers that could complement the minimal panel proposed for the in-vitro MSC definition, being shared and strongly expressed by BM- and CB-derived MSC. We have also identified CD143 as a marker exclusively expressed on MSC derived from adult tissue sources. Further studies will elucidate the biological role of CD143 and its potential association with tissue-specific MSC features.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0755-3) contains supplementary material, which is available to authorized users.
BackgroundIncreasing evidence suggests the safety and efficacy of mesenchymal stromal cells (MSC) as advanced therapy medicinal products because of their immunomodulatory properties and supportive role in hematopoiesis. Although bone marrow remains the most common source for obtaining off-the-shelf MSC, cord blood (CB) represents an alternative source, which can be collected noninvasively and without major ethical concerns. However, the low estimated frequency and inconsistency of successful isolation represent open challenges for the use of CB-derived MSC in clinical trials. This study explores whether CB may represent a suitable source of MSC for clinical use and analyzes several in vitro parameters useful to better define the quality of CB-derived MSC prior to clinical application.MethodsCB units (n = 50) selected according to quality criteria (CB volume ≥ 20 ml, time from collection ≤ 24 h) were cultured using a standardized procedure for CB-MSC generation. MSC were analyzed for their growth potential and secondary colony-forming capacity. Immunophenotype and multilineage differentiation potential of culture-expanded CB-MSC were assessed to verify MSC identity. The immunomodulatory activity at resting conditions and after inflammatory priming (IFN-γ-1b and TNF-α for 48 hours) was explored to assess the in vitro potency of CB-MSC prior to clinical application. Molecular karyotyping was used to assess the genetic stability after prolonged MSC expansion.ResultsWe were able to isolate MSC colonies from 44% of the processed units. Our results do not support a role of CB volume in determining the outcome of the cultures, in terms of both isolation and proliferative capacity of CB-MSC. Particularly, we have confirmed the existence of two different CB-MSC populations named short- and long-living (SL- and LL-) CBMSC, clearly diverging in their growth capacity and secondary colony-forming efficiency. Only LL-CBMSC were able to expand consistently and to survive for longer periods in vitro, while preserving genetic stability. Therefore, they may represent interesting candidates for therapeutic applications. We have also observed that LL-CBMSC were not equally immunosuppressive, particularly after inflammatory priming and despite upregulating priming-inducible markers.ConclusionsThis work supports the use of CB as a potential MSC source for clinical applications, remaining more readily available compared to conventional sources. We have provided evidence that not all LL-CBMSC are equally immunosuppressive in an inflammatory environment, suggesting the need to include the assessment of potency among the release criteria for each CB-MSC batch intended for clinical use, at least for the treatment of immune disorders as GvHD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0465-2) contains supplementary material, which is available to authorized users.
ObjectivesGestational Trophoblastic Disease (GTD) comprises a group of disorders that derive from the placenta. Even if full recovery is generally expected, women diagnosed with GTD have to confront: the loss of a pregnancy, a potentially life-threatening diagnosis and delays in future pregnancies. The aim of the study is to evaluate the psychological impact of GTD, focusing on perceived fertility, depression and anxiety.Methods37 patients treated for GTD at San Raffaele Hospital, Milan, took part in the study. The STAI-Y (State-Trait Anxiety Inventory), the BDI-SF (Beck Depression Scale-Short Form) and the FPI (Fertility Problem Inventory) were used. Patients were grouped on the basis of presence of children (with or without), age (< or ≥35) and type of diagnosis (Hydatidiform Mole, HM, or Gestational Trophoblastic Neoplasia, GTN). Differences in the values between variables were assessed by a t-type test statistic. Three-way ANOVAs were also carried out considering the same block factors.ResultsThe study highlights that women suffering from GTN had higher depression scores compared to women suffering from HM. A significant correlation was found between anxiety (state and trait) and depression. Younger women presented higher Global Stress scores on the FPI, especially tied to Need for Parenthood and Relationship Concern subscales. Need for Parenthood mean scores significantly varied between women with and without children too.ConclusionsWe can conclude that fertility perception seems to be negatively affected by GTD diagnosis, particularly in younger women and in those without children. Patients should be followed by a multidisciplinary team so as to be supported in the disease’s psychological aspects too.
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