The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cells in vitro.

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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.

Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P<0.0001). At univariate analysis LAIR1(+) was significantly associated with longer time to first treatment (P=0.0002). This favorable effect of LAIR1(+) was confirmed by multivariate analysis (hazard ratio=2.1, P=0.03 for LAIR1). Our results indicate that LAIR1 expression is a reliable and inexpensive marker capable of independently predicting time to first treatment in newly diagnosed unselected patients with chronic lymphocytic leukemia.

High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources.

BACKGROUND: Mesenchymal 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. METHODS: BM-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. RESULTS: Based 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. CONCLUSIONS: This 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.

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells.

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4-64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

BACKGROUND: Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). MATERIALS AND METHODS: We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. RESULTS: After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. DISCUSSION: We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

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

BACKGROUND: Increasing 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. METHODS: CB 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. RESULTS: We 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. CONCLUSIONS: This 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.

Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

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.

Heterogeneity of Specific CD4+ and CD8+ T Cells Stimulated by CMV pp65 and IE1 Antigens.

Characterization of human cytomegalovirus-specific T cells (CMV-T) is of critical importance for their potential use in adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation. Background frequencies of CMV-T in peripheral blood mononuclear cells (PBMCs) of CMV-seropositive healthy subjects are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition has sometimes been neglected or difficult to assess in these settings. We explored the identity and the functionality of pp65-specific and IE1-specific T cells, enriched in short-term cultures from PBMCs. Antigen-specific T cells were further isolated by IFN-γ capture system and/or CD154 microbeads. Frequency of IE1-specific cytotoxic T cells in PBMCs secreting IFN-γ was higher compared with the pp65-specific one, whereas the latter cell types showed a higher median CD107a degranulation. Cell viability, rate of CMV-T increase, and multicytokine secretion profile after epitope-specific short-term cultures were heterogenous. T cells were mainly of late effector stages but they significantly dropped off upon CMV rechallenge with peptide pools. In parallel, CMV-T expansion was accompanied by a significant increase of cytotoxic naive/memory stem cells (CTLs), whereas the CD4 counterpart significantly increased only upon stimulation with IE1. Outcome was variable and showed donor and epitope dependency. Differences in human leukocyte antigen and epitope dominance and variability in the relative number of CD3 effector cells and IFN-γ/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching adoptive immunotherapy.