Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105+ cells.

Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long-term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM-CD105(+) cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 × 10(5) ± 0.9 × 10(5) CD105(+) cells from 10 small volume (10-25 ml) BM samples achieving an enrichment >100-fold in MSC. Seeding 2 × 10(3) immunoselected cells/cm(2) we were able to produce 2.5 × 10(8) ± 0.7 × 10(8) MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29(+), CD90(+), CD73(+), CD105(+), CD45(-); they suppressed allogeneic T-cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM-CD105(+) cells.

Low usage rate of banked sibling cord blood units in hematopoietic stem cell transplantation for children with hematological malignancies: implications for directed cord blood banking policies.

Directed sibling cord blood banking is indicated in women delivering healthy babies who already have a sibling with a disease that is potentially treatable with an allogeneic cord blood transplant. We evaluated the effectiveness of a national directed cord blood banking program in sibling HLA-identical stem cell transplantation for hematological malignancies and the factors influencing the usage rate of the stored cord blood units. Fifty families were enrolled from which, 48 cord blood units were successfully collected and 2 collections failed due to damaged cord/placenta at delivery. Among enrolled families 4 children needed transplantation; however, only one was successfully transplanted using the collected cord blood unit containing 2×10(7) nucleated cells/kg in conjunction with a small volume of bone marrow from the same HLA-identical donor. Two children received grafts from matched unrelated donors because their sibling cord blood was HLA-haploidentical, while the fourth one received bone marrow from his HLA-identical brother, since cord blood could not be collected due to damaged cord/placenta at delivery. With a median follow-up of 6 years (range, 2-12) for the 9 remaining HLA-matched cord blood units, none from the prospective recipients needed transplantation. The low utilization rate of sibling cord blood in the setting of hematopoietic stem cell transplantation for pediatric hematological malignant diseases necessitates the development of directed cord blood banking programs that limit long-term storage for banked cord blood units with low probability of usage such as non-HLA-identical or identical to patients who are in long-term complete remission.

Directed sibling donor cord blood banking for children with beta-thalassemia major in Greece: usage rate and outcome of transplantation for HLA-matched units.

Several cord blood banks store cord blood units from healthy siblings of patients, who are candidates for stem cell transplantation. We analyzed the quality characteristics of 50 cord blood units collected from families with beta-thalassemia major and the outcome of subsequent stem cell transplantations during a 15-year period. All cord blood units were found suitable for banking based on a minimum net volume of 40 ml. The mean volume of the units was 98.9 ml; the mean total nucleated cell count (NC) was 7.8 x 10(8) and the mean CD34+ cell count was 2.8 x 10(6). Eight out of twelve HLA matched collections were released for transplantation. All but one recipient belonged to Pesaro II-III risk classes. Three patients received a cord blood graft with >5 x 10(7) NC/kg . One of them with Pesaro class I disease engrafted, whereas the other two who failed to engraft, were re-transplanted with bone marrow from the same donor later. Cord blood grafts containing NCs <4 x 10(7)/kg combined with reduced volume bone marrow from the same donor were used in all 5 remaining cases and stable engraftment was achieved. All patients survived, 7/8 thalassemia-free. Cord blood banking from healthy siblings of children with beta-thalassemia major can result in a successful transplantation in cases in which there is HLA compatibility. However, in high-risk patients, the use of combined cord blood and bone marrow grafts seems necessary in order to ensure stable engraftment, especially when cord blood unit cell counts are low.

Distinct roles of TLR4 and CD14 in LPS-induced inflammatory responses of neonates.

During infections, pathogens bind to toll-like receptor (TLR)4 and CD14 receptors and induce cytokine release, leading to inflammation. Here, we investigated TLR4 and CD14 expression on peripheral blood leukocytes (PBLs) and their roles in lipopolysaccharide (LPS)-induced cytokine and chemokine release. Full-term and preterm neonates and adults were studied. PBLs were pretreated with anti-TLR4- and anti-CD14-blocking antibodies and stimulated with LPS. Cytokine and chemokine levels were measured in supernatants. TLR4, CD14 expression, and LPS-induced CXCL8 release were higher in neonates, possibly contributing to aberrant inflammation. TLR4 blockade resulted in approximately 3-fold greater suppression of LPS-induced CXCL8 release in preterm neonates (38%) than in adults (14%). CD14 blockade (approximately 80%) in neonates induced approximately 3-fold greater inhibition of CXCL8 release, compared with anti-TLR4 (approximately 30%). Anti-TLR4 partly (50-60%) inhibited IL-10 and TNF-alpha, whereas anti-CD14 completely suppressed their release. Our findings reveal that neonates depend more on TLR4 for CXCL8 release. Furthermore, neonatal LPS-induced CXCL8 release, apart from TLR4/CD14-mediated signaling, is regulated by LPS interactions with other TLRs and/or immune receptors. IL-10 and TNF-alpha release depends on LPS binding not only to CD14/TLR4 but also to CD14 associated with another TLR. Our findings reveal the contribution of TLR4 and CD14 in neonatal cytokine and chemokine release and could aid in design of antagonists to prevent harmful inflammation.

Lack of Fas (APO-1/CD95) gene structural alterations or transcript variant ratio changes in breast cancer.

Fas (APO-1/CD95) is a transmembrane receptor protein involved in cell death signaling. Fas receptor and ligand are both expressed in breast cancer cells, however these cells are resistant to apoptosis. Fas gene mutations were detected in hematological and solid tumors, while overexpression of a soluble Fas isoform in serum was related to cancer stage and prognosis. In this work, direct sequencing of exons 6 and 9 of the Fas gene from 90 patients did not reveal any structural alterations. Moreover, no decrease was found in the ratio of the corresponding mRNA species of transmembrane versus soluble Fas isoforms in 31 breast cancer samples compared to 14 controls. Therefore, inhibition of Fas-mediated apoptosis may not be due to structural alterations in the critical exons 6 and 9 of the Fas gene or a shift of expression towards the soluble Fas isoform, but to other mechanisms operating in breast cancer cells.

Detection of herpes simplex virus, cytomegalovirus, and Epstein-Barr virus in the semen of men attending an infertility clinic.

OBJECTIVE: To investigate the prevalence of herpes simplex virus (HSV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in the semen of men with fertility problems. DESIGN: A descriptive clinical study. SETTINGS: Outpatient infertility clinic of a private hospital. PATIENT(S): One hundred thirteen men attending an infertility clinic in Athens. INTERVENTION(S): Semen samples were collected by masturbation. MAIN OUTCOME MEASURE(S): Detection of HSV, CMV, and EBV in semen by a nested polymerase chain reaction technique. Complete spermogram including sperm count, motility, pH, viscosity, and morphology. RESULT(S): Viral DNA was detected by the nested polymerase chain reaction technique in 64 (56.6%) of 113 semen samples. Specifically, HSV DNA was detected in 56 (49.5%) semen samples, EBV DNA in 19 (16.8%) semen samples, and CMV DNA in 8 (7.1%) semen samples. HSV was significantly related to low sperm count and poor motility. In contrast, CMV and EBV did not show any association with sperm concentration and motility. CONCLUSION(S): Herpes simplex virus seems to play a significant role in male infertility, and its early detection by the nested polymerase chain reaction technique will permit successful antiviral therapy to increase the possibility for fertility restoration and long-term protection of the sperm quality. Finally, the detection of herpes viruses within semen will allow better control of the transmission of these viruses.

Mesenchymal derivatives of genetically unstable human embryonic stem cells are maintained unstable but undergo senescence in culture as do bone marrow-derived mesenchymal stem cells.

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here, we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages, respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations, suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs, which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells.

Myofibroblasts generated in culture from sclerotic skin lesions of a patient with extensive chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation are of recipient origin.

Abstract The origin (recipient/donor) of the myofibroblasts mediating fibrosis in sclerodermatous chronic graft-versus-host disease (cGvHD) was investigated. Sclerodermatous specimens obtained from a patient with extensive cGvHD after an HLA-identical sibling bone marrow transplantation were cultured in order to derive tissue myofibroblasts. All proliferating a-SMA+ fibroblastoid cells revealed recipient origin as examined by variable number tandem repeat (VNTR)-PCR. This case report shows that fibrosis in sclerodermatous lesions results from the activation and proliferation of locally-derived recipient fibroblasts rather than from donor-derived fibroblasts or circulating fibrocytes.

Inflammatory chemokine expression in the peripheral blood of neonates with perinatal asphyxia and perinatal or nosocomial infections.

AIM: The inflammatory response induced by perinatal infections and asphyxia is considered to participate in neonatal brain damage. Inflammatory responses are characterized by the expression of chemokines. Although chemokine levels have been investigated in healthy newborns, their role during neonatal pathological conditions has not been studied. The aim of our study was to examine chemokine serum levels in asphyxiated and infected neonates. METHODS: Peripheral blood samples were obtained from perinatally asphyxiated and infected neonates during the first days of life and from neonates who developed nosocomial infections. Serum levels of interleukin-8 (IL-8), interferon-gamma-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and regulated upon activation, normal T cells expressed and secreted (RANTES) were determined. RESULTS: In perinatally asphyxiated neonates, IL-8 levels were significantly elevated on the 1st day of life. In perinatally infected neonates, IL-8 and IP-10 levels were significantly increased on the 1st day of life, while RANTES levels were significantly lower and remained so until the 4th day. In nosocomially infected neonates, IL-8, IP-10 and MIP-1alpha levels were significantly increased on diagnosis of infection. CONCLUSION: The neonatal immune system is able to produce chemokines for the induction of an inflammatory response during perinatal asphyxia and perinatal or nosocomial infections. Blockade of inflammatory chemokines could possibly contribute to the prevention of brain damage.