Chryssoula Perdikogianni

Study of oncogenic transformation in ex vivo expanded mesenchymal cells, from paediatric bone marrow

Abstract.  Objectives: Mesenchymal stromal cells (MSCs) have attracted considerable interest in both the scientific and clinical fields. In order to obtain a sufficient cell number for application, their in vitro expansion is necessary, but during this process their characteristics may be altered and cells may acquire oncogenic properties. We have investigated properties of MSC that may be related to oncogenesis, a critical parameter that has to be evaluated prior to MSC clinical use. Materials and methods: We studied the expression of p53, p16, RB, H‐RAS and human telomerase reverse transcriptase (hTERT) in MSCs from bone marrow of children diagnosed with idiopathic thrombocytopenic purpura (ITP) and autoimmune neutropenia. The same cells were seeded in soft agar to confirm their anchorage dependence and were karyotypically analysed. Finally, MSCs were subcutaneously transplanted into SCID mice and their ectopic osteogenic as well as tumorigenic potential was evaluated. Results: We have shown that MSCs derived from bone marrow of children with ITP and autoimmune neutropenia do not undergo transformation, the cells expressed normal levels of p53, p16, RB and H‐RAS. Expression of hTERT was undetectable, chromosome content remained stable, and their anchorage dependence was confirmed. In an in vivo model, when MSCs were subcutaneously transplanted into SCID mice, no tumorigenesis was observed. Conclusions: These findings suggest that MSCs from bone marrow of children do not have oncogenic properties and, therefore, represent validate candidates for applications in regenerative medicine.

Could cord blood be a source of mesenchymal stromal cells for clinical use

BACKGROUND Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. METHODS Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated. RESULTS Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. DISCUSSION These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.

Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases

BACKGROUND Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. METHODS BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. RESULTS MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. DISCUSSION MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

The impact of mode of delivery and gestational age on cord blood hematopoietic stem/progenitor cells.

Human cord blood has been successfully used as an alternative source of hematopoietic stem cells suitable for transplantation. The aim of this study was to assess the impact of gestational age and the mode of delivery on cord blood hematopoietic stem/progenitor cell characteristics. The mode of delivery does not seem to affect either the replating capacity of hematopoietic progenitors colony-forming unit-granulocyte-macrophage or the cord blood content in CD34+ cells. The higher percentage of CD34+ cells in cord blood from preterm deliveries compared to full-term ones indicates that hematopoietic progenitors from preterm cord blood may be suitable for transplantation. These findings should be taken into consideration when selection of cord blood units is required for potential use in transplantation.

Idiopathic thrombocytopenic purpura in childhood: twenty years of experience in a single center.

Background:  Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder with a variable clinical course.

Childhood Yersinia enterocolitica infection in Crete.

Yersinia enterocolitica appears to be a common cause of enteritis in humans, yet great variations have been noted among countries and the epidemiology of the infection is poorly understood [1–7]. Yersiniosis occurs either sporadically or as outbreaks often attributable to contaminated pork, water, milk and bean sprouts [2, 4, 8]. Individuals with a predisposing underlying condition including immunosuppression and iron overload are more prone to systemic disease, but normal hosts and especially infants can also be affected [2]. In order to investigate childhood Y. enterocolitica infection in Crete over a long period of time, we reviewed the records of all outpatients and inpatients with a stool culture positive for Yersinia at the University General Hospital of Heraklion from January 1993 through December 2004. The hospital is the major health center in Crete, serving a population of 611,000, of whom 132,000 are children aged less than 14 years (2001 census data). All fecal samples were routinely processed for Salmonella spp., Shigella spp., Yersinia spp., Aeromonas spp. and Campylobacter spp. MacConkey, Salmonella-Shigella, Hektoen enteric, cefsulodin–Irgasan–novobiocin (CIN) and campylosel agar plates were used (bioMérieux, Marcy L’ Etoile, France). Plates were incubated for 48 h at 35°C, except for campylosel agar plates, which were incubated at 42°C under microaerophilic conditions, and CIN agar plates, which were incubated at 25°C. For Yersinia enrichment, a portion of stool was inoculated onto 10 ml of phosphate-buffered saline (Bio-Rad, Marnes-la-Coquette, France), incubated at 4°C for 3 weeks and subcultured in CIN agar on a weekly basis. Both CIN agar and cold enrichment procedure were utilized for recovery of Yersinia throughout the study period. Identification of the isolates was performed using standard methods such as the API 20E system (bioMérieux) and the automated Vitek 2 system (bioMérieux). Serotyping was performed using commercial antisera with the slide-agglutination method (Bio-Rad). Antimicrobial susceptibility testing was performed using the disk diffusion method recommended by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) [9]. Statistical analysis was performed using 95% confidence intervals for estimation of relative risk and the chi-square test for estimation of significance. During the 12-year study period, Y. enterocolitica accounted for 5.52% (71 of 1,286) of all enteric pathogens in children and for 2.02% (22 of 1,091) in adults, being thus more common among children than among adults (relative risk, 2.74; 95% confidence interval, 1.71–4.39; p<0.0001). The male:female ratio was 1.09:1. Among the 71 children, 27 (38%) were managed as outpatients and 44 (62%) were hospitalized. Y. enterocolitica cases were observed throughout the study period (annual range, 3–13 cases). Among the 71 total cases, 32 (45%) occurred in December and January (Fig. 1). Clear evidence of exposure to potential sources of contamination was not elicited. All Yersinia isolates were found to be Y. enterocolitica serogroup O:3. Campylobacter spp. was concomitantly isolated in one patient. In three cases there was an underlying condition requiring overtransfusion: two were β-thalassemia patients and one child suffered from Diamond-Blackfan anemia. No case of bacteremia, septicemia or extraintestinal complications was documented throughout the study period. All Yersinia enterocolitica strains grew on routine stool culture media. Among the 71 isolates, 59 (83%) were recovered by direct plating, and nine (12.7%) and three (4.22%) were recovered by 1and 2-weeks of cold enrichment, respectively. All 71 isolates were susceptible to third-generation cephalosporins, gentamicin, tobramycin and the quinolones, and all but one to trimethoprimE. Galanakis (*) . C. Perdikogianni . E. Giannoussi . M. Kalmanti Department of Paediatrics, University of Crete, P.O. Box 2208, Heraklion, 71003, Greece e-mail: egalanak@med.uoc.gr Tel.: +30-2810-392012 Fax: +30-2810-392827

Expression levels of ASNS in mesenchymal stromal cells in childhood acute lymphoblastic leukemia

Increased levels of asparagine synthetase (ASNS), an enzyme producing intracellular asparagine, have been implicated in the development of asparaginase resistance. The aim of this study was to assess ASNS mRNA and protein expression in bone marrow cell populations of children with acute lymphoblastic leukemia (ALL). Bone marrow mononuclear cells at diagnosis, day 33 of treatment, and after completion of chemotherapy were isolated and studied. ASNS mRNA expression was assessed by real-time PCR, and protein levels by Western blot. Our results indicate that MSC ASNS mRNA expression is upregulated in ALL samples compared to controls. ASNS expression of mesenchymal stromal cells (MSC) was found to be 2.3 times higher than that of blasts at diagnosis of ALL. We also observed that the values of the ASNS mRNA of MSC seem to reach a peak at diagnosis, and tend to decline with treatment. No correlation was found between the ASNS mRNA and protein levels. Chemotherapy does not exert any effect on the protein expression. Variability of asparaginase-induced effect may be attributable to factors involved in the interaction of hematopoietic cells with their microenvironment.

Non-tuberculous mycobacterial cervical lymphadenitis in the immunocompetent child: diagnostic and treatment approach

Non-tuberculous mycobacterial disease in the otherwise well child commonly presents as a localized, slow-progressing, cervicofacial lymphadenopathy. Despite recent advances, several aspects of the susceptibility to and the course of the disease remain to be better understood, and diagnosis and management of non-tuberculous mycobacterial lymphadenitis in children are often controversial. Differential diagnosis should include other infections, in particular tuberculosis, local cysts and malignancies. In the majority of cases, untreated disease progresses to spontaneous drainage, fistula formation and scarring and a high index of clinical suspicion is necessary for timely diagnosis and treatment. Excisional surgery seems to remain the gold standard. Medical treatment might be considered as an appropriate alternative in case there is a high risk of surgical complications or in case surgery has already been unsuccessful. Family reassurance and the watch-and-wait approach may be an option in milder disease.

IN VITRO ASSESSMENT OF MESENCHYMAL STROMAL CELL CHARACTERISTICS: IMPLICATIONS FOR THEIR CLINICAL USE

INTRODUCTION: Bone marrow (BM) stroma represents a source of progenitor stromal cells, termed mesenchymal stromal cells (MSCs), which are multipotent and can differentiate into cartilage, bone, and adipose tissue. Several questions have arisen regarding their long-term expansion and their safety before use. OBJECTIVE: Our goal was to assess the long-term expansion and safety of MSCs in clinical practice. METHODS: MSCs from BM of children with benign hematologic disorders and solid tumors without BM involvement were isolated and cultured for 10 consecutive passages (P). Immunophenotypic and functional characteristics, apoptosis, and the expression of cell cycle regulatory genes (p53, p16, and Rb) and signal transduction genes (H-Ras) involved in oncogenesis were assessed. RESULTS: MSCs expressed mesenchymal-related surface antigens, >85% from P1. They had the ability to differentiate into osteocytes, adipocytes, and chondrocytes (reverse-transcription polymerase chain reaction). Colony forming units (fibroblast) ranged from 40.71 ± 4.3 at P1 to 15.5 ± 6.7 at P10. Their doubling time was 2.01 ± 0.14 days at P1 and 3.5 ± 1.19 days at P9. A low percentage of apoptotic cells was detected (7-amino-actinomycin D [7AAD]) at P2 until P10. MSCs were resistant to apoptosis under serum-deprivation conditions. The expression of the cell cycle genes studied was not statistically different compared with controls, and cells did not grow on soft agar. CONCLUSIONS: MSCs isolated from BM of children retain their characteristics for a serial number of passages and survive under serum-deprivation conditions, a necessary process in a transplantation setting. The cells do not have oncogenic properties, as shown by normal expression levels of oncogenes and tumor suppressor genes, and no growth on soft agar. These findings enhance the use of MSCs in clinical applications.