Angela Cometa

Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-term In vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms

Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell‐therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet‐lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell‐therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL‐MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune‐regulatory effect of MSCs on the alloantigen‐specific immune response in mixed lymphocyte culture (MLC). We found that MSCs‐PL are comparable to MSCs‐FCS in their capacity to: (i) decrease alloantigen‐induced cytotoxic activity; (ii) favor differentiation of CD4+ T‐cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL‐10 in MLC supernatant, as well as induce a striking augmentation of IL‐6 production. As compared with MSCs‐PL, MSCs‐FCS were more efficient in suppressing alloantigen‐induced lymphocyte subset proliferation and reducing early IFNγ‐secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs‐PL can be used as an alternative to MSCs‐FCS, although these latter cells might be more suitable for preventing/treating alloreactivity‐related immune complications. J. Cell. Physiol. 211: 121–130, 2007.

Co-infusion of ex vivo- expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.

Polyomavirus BK-specific immunity after kidney transplantation.

Failure to mount or maintain a protective immune response may influence the development of polyomavirus BK (BKV)-associated nephropathy (PVAN). However, limited data are so far available on BKV-specific immunity after kidney transplantation. BKV-specific cellular immune response was retrospectively analyzed in kidney recipients with or without BKV infection/reactivation by measuring the frequency of interferon (IFN)-gamma-secreting cells in peripheral blood. Patients with BKV-active infection and good renal function (n=6) had a mean BKV-specific lymphocyte frequency 2 log lower than healthy controls and in the same range as BKV-seropositive recipients without active infection (n=7). Patients with PVAN (n=5) revealed undetectable levels of BKV-specific cells. However, two patients from the latter cohort treated with immunosuppression reduction showed the emergence of specific immunity, with IFN-gamma production in the same range as healthy controls. Our preliminary data suggest that lack of protective immunity toward BKV may favor the occurrence of BKV active infection and influence the progression to PVAN.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

Umbilical cord blood is an attractive source of stem cells for several cell-based therapies. In this paper, it is shown that umbilical cord blood-derived mesenchymal stroma cells, cultured in the presence of platelet lysate, have an increased proliferative potential but comparable immunomodulatory functions relative to their bone marrow-derived counterparts. Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

Ex vivo expansion of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed.

Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease

BACKGROUND AIMS Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohns disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application. METHODS MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD). RESULTS MSC were successfully expanded from all patients. Colony-forming unit-fibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [12-84% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens. CONCLUSIONS CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease.

Human adipose-derived stem cells (hASCs) proliferate and differentiate in osteoblast-like cells on trabecular titanium scaffolds

The use of stem cells in regenerative medicine is an appealing area of research that has received a great deal of interest in recent years. The population called human adipose tissue-derived stem cells (hASCs) share many of the characteristic of its counterpart of marrow including extensive proliferative potential and the ability to undergo multilineage differentiation along classical mesenchymal lineages: adipogenesis, chondrogenesis, osteogenesis, and myogenesis. The aim of this study was to evaluate with biochemical and morphological methods the adhesion and differentiation of hASCs grown on trabecular titanium scaffolds. The hASCs isolated from subcutaneous adipose tissue after digestion with collagenase were seeded on monolayer and on trabecular titanium scaffolds and incubated at 37 degrees C in 5% CO(2) with osteogenic medium or control medium.The results showed that hASCs were able to adhere to titanium scaffolds, to proliferate, to acquire an osteoblastic-like phenotype, and to produce a calcified extracellular matrix with protein, such as, decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type I collagen. These data suggest that this kind of scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue.

Variations of the perforin gene in patients with multiple sclerosis

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13–3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10–1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Different molecular mechanisms causing 9p21 deletions in acute lymphoblastic leukemia of childhood

Deletion of chromosome 9p21 is a crucial event for the development of several cancers including acute lymphoblastic leukemia (ALL). Double strand breaks (DSBs) triggering 9p21 deletions in ALL have been reported to occur at a few defined sites by illegitimate action of the V(D)J recombination activating protein complex. We have cloned 23 breakpoint junctions for a total of 46 breakpoints in 17 childhood ALL (9 B- and 8 T-lineages) showing different size deletions at one or both homologous chromosomes 9 to investigate which particular sequences make the region susceptible to interstitial deletion. We found that half of 9p21 deletion breakpoints were mediated by ectopic V(D)J recombination mechanisms whereas the remaining half were associated to repeated sequences, including some with potential for non-B DNA structure formation. Other mechanisms, such as microhomology-mediated repair, that are common in other cancers, play only a very minor role in ALL. Nucleotide insertions at breakpoint junctions and microinversions flanking the breakpoints have been detected at 20/23 and 2/23 breakpoint junctions, respectively, both in the presence of recombination signal sequence (RSS)-like sequences and of other unspecific sequences. The majority of breakpoints were unique except for two cases, both T-ALL, showing identical deletions. Four of the 46 breakpoints coincide with those reported in other cases, thus confirming the presence of recurrent deletion hotspots. Among the six cases with heterozygous 9p deletions, we found that the remaining CDKN2A and CDKN2B alleles were hypermethylated at CpG islands.