RESUMO
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.
Assuntos
Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Imunofenotipagem/métodos , Células-Tronco Mesenquimais/citologia , Receptores de Superfície Celular/metabolismo , Adulto , Diferenciação Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Criança , Hibridização Genômica Comparativa , Endoglina , Humanos , Teste de Cultura Mista de Linfócitos , Linfócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/estatística & dados numéricos , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Bancos de Sangue , Criança , Família , Feminino , Sangue Fetal , Feto , Grécia , Antígenos HLA/imunologia , Neoplasias Hematológicas/terapia , Histocompatibilidade/imunologia , Humanos , Lactente , Masculino , Gravidez , Irmãos , Doadores de Tecidos/estatística & dados numéricosRESUMO
We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor.
Assuntos
Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Teste de Histocompatibilidade , Diagnóstico Pré-Implantação , Irmãos , Plaquetas/citologia , Células da Medula Óssea/citologia , Contagem de Células , Pré-Escolar , Embrião de Mamíferos/imunologia , Feminino , Fertilização in vitro , Sangue Fetal/citologia , Sobrevivência de Enxerto , Doença Granulomatosa Crônica/genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação de Sentido Incorreto/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Explosão Respiratória/efeitos dos fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Quimeras de Transplante/genética , Quimeras de Transplante/metabolismo , Resultado do TratamentoRESUMO
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.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Antígenos HLA/imunologia , Talassemia beta/terapia , Adolescente , Bancos de Sangue , Criança , Pré-Escolar , Grécia , Humanos , Irmãos , Doadores de Tecidos , Resultado do Tratamento , Talassemia beta/cirurgiaRESUMO
BACKGROUND: Exercise in humans augments the mobilization of circulating hematopoietic progenitor cells (CD34(+)) from the bone marrow. We investigated the effect of inflammation on erythroid marrow activity by mobilization of erythroid progenitor cells (EPs) along with soluble markers of erythropoiesis. METHODS: Ten healthy athletes who participated in an ultradistance foot race participated in the study. Peripheral blood mononuclear cells were isolated, before (phase I), at the end (phase II), and at 48 h post-race (phase III). EPs were detected as burst colony forming units (BFU-e) and colonies were scored at day 14. Markers of inflammation (C-reactive protein, serum amyloid-A, interleukin-6, ferritin and S100B) and bone marrow activity (erythropoietin, soluble transferrin receptor and lipocalin-2) were assessed. RESULTS: An approximately three-fold decrease in BFU-e number was observed at phase II. sTfR concentrations were also decreased at phase II and remained decreased at phase III. However, EPO and lipocalin-2 concentrations reached a maximum value at phase II, with a tendency to decrease at phase III. CONCLUSIONS: These findings indicate that exercise-induced inflammation modulates bone marrow homeostasis leading to an increase in leukocyte turnover and a decrease in erythroid compartment. It appears that lipocalin-2 is the main factor that regulates the production and mobilization of EPs.
Assuntos
Biomarcadores/sangue , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Eritropoese/fisiologia , Inflamação/sangue , Esforço Físico/fisiologia , Corrida/lesões , Proteínas de Fase Aguda/imunologia , Proteínas de Fase Aguda/metabolismo , Atletas , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Precursoras Eritroides/imunologia , Eritropoese/imunologia , Eritropoetina/sangue , Ferritinas/sangue , Humanos , Inflamação/etiologia , Inflamação/imunologia , Lipocalina-2 , Lipocalinas/imunologia , Lipocalinas/metabolismo , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores da Transferrina/sangue , Fatores de TempoRESUMO
Endothelial progenitor cells (EPCs) and the recently described circulating fibrocytes (CFs) are strongly associated with tissue repair. We investigated the kinetics of both "repair" progenitor cells in healthy athletes who participated in the "Spartahlon" ultradistance foot race (246 km continuous running exercise), which provides a unique model of inducing dramatic systemic inflammatory changes. Peripheral blood mononuclear cells (PBMCs) were isolated from 10 volunteer athletes, who completed successfully the race, before, at the end, and at 48 h post-race. EPCs and CFs were detected as endothelial colony-forming units (CFU-ECs) and as the number of adherent with a spindle-shaped morphology Collagen I(+) cells detected after 6-day culture of PBMCs, respectively. The marked increase of plasma levels of CRP, IL-6, SAA, MCP-1, IL-8, sVCAM-1, sICAM-1, thrombomodulin (sTM) and NT-pro-BNP at the end of race established acute inflammation and tissue injury. EPCs increased by nearly eleven-fold in peripheral blood at the end of the race from 44.5+/-2.5/ml to 494.6+/-27.9/ml and remained increased 428.5+/-31.5/ml at 48 h post-race (p<0.0001). The number of the fibrocytes cultured from PBMCs obtained before, at the end, and 48 h post-race did not reveal any significant difference. These findings indicate that bone marrow responses to acute inflammatory damage, induced by exhausting exercise, with a rapid release of EPCs but not CFs into circulation. Given the ability of EPCs to promote angiogenesis and vascular regeneration, we may suggest that this kind of cell mobilization may serve as a physiologic repair mechanism in acute inflammatory tissue injury.
Assuntos
Inflamação/sangue , Células-Tronco Mesenquimais/fisiologia , Regeneração/fisiologia , Corrida/lesões , Adulto , Antígenos de Diferenciação/análise , Biomarcadores , Contagem de Células Sanguíneas , Medula Óssea/fisiopatologia , Moléculas de Adesão Celular/sangue , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Células Endoteliais/patologia , Humanos , Inflamação/etiologia , Inflamação/fisiopatologia , Mediadores da Inflamação/sangue , Masculino , Células-Tronco Mesenquimais/química , Pessoa de Meia-Idade , Monócitos/patologia , Miosite/sangue , Miosite/etiologia , Miosite/fisiopatologia , Neovascularização Fisiológica , Corrida/fisiologiaRESUMO
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.