RESUMEN
Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T cells transduced with a second-generation (4.1BB) CD19-directed CAR for treatment of patients with BCP-ALL in a hospital-exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR-Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy-based manufacturing process (CD19-CAR-Lenti_ALLO). Thirteen children/young adults received ALLO-CAR-T cells between March 2021 and October 2022. Doses ranged between 1.0 × 106 and 3.0 × 106 CAR-T cells per kg. The toxicity profile was comparable with that of autologous CAR-T cells, characterized mainly by cytopenia, cytokine release syndrome (maximum grade 1), and grade 2 immune-effector cell-associated neurotoxicity syndrome. One case of acute graft-versus-host disease (GVHD) occurred and was rapidly controlled with steroids and ruxolitinib. None of the other patients, including 3 given ALLO-CAR-T cells from an HLA-haploidentical donor, experienced GVHD. Two patients received ALLO-CAR-T cells before HSCT and showed a significant expansion of CAR-T cells without any sign of GVHD. All patients obtained complete remission (CR) with absence of minimal residual disease in the bone marrow. With a median follow-up of 12 months (range, 5-21), 8 of 13 patients maintained CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly refractory BCP-ALL relapsing after allo-HSCT without showing increased toxicity as compared with autologous CAR-T cells.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto Joven , Humanos , Niño , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linfocitos T , Enfermedad Injerto contra Huésped/etiología , Inmunoterapia Adoptiva/efectos adversos , Antígenos CD19RESUMEN
BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. METHODS: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). RESULTS: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. CONCLUSIONS: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).
Asunto(s)
Inmunoterapia Adoptiva , Neuroblastoma , Receptores Quiméricos de Antígenos , Niño , Humanos , Caspasa 9/efectos adversos , Caspasa 9/genética , Caspasa 9/metabolismo , Caspasa 9/uso terapéutico , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/terapia , Neuroblastoma/genética , Neuroblastoma/terapia , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
Previous studies have shown that human liver stem-like cells (HLSCs) may undergo differentiation in vitro into urea producing hepatocytes and in vivo may sustain liver function in models of experimentally induced acute liver injury. The aim of this study was to assess the safety of HLSCs intrahepatic administration in inherited neonatal-onset hyperammonemia. The study was approved by the Agenzia Italiana del Farmaco on favorable opinion of the Italian Institute of Health as an open-label, prospective, uncontrolled, monocentric Phase I study (HLSC 01-11, EudraCT-No. 2012-002120-33). Three patients affected by argininosuccinic aciduria (patient 1) and methylmalonic acidemia (patients 2 and 3) and included in the liver transplantation list were enrolled. In all patients, HLSCs were administered by percutaneous intrahepatic injections (once a week for two consecutive weeks) within the first months of life. The first patient received 125,000 HLSCs x gram of liver/dose while the other two patients received twice this dose. No immunosuppression was administered since HLSCs possess immunomodulatory activities. None of the patients experienced infections, hyperammonemia decompensation, or other adverse events during the whole observation period. No donor specific antibodies (DSA) against HLSCs were detected. Patients were metabolic stable despite an increase (~30%) in protein intake. Two patients underwent liver transplantation after 19 and 11 months respectively, and after explantation, the native livers showed no histological alterations. In conclusion, percutaneous intrahepatic administration of HLSCs was safe in newborn with inherited neonatal-onset hyperammonemia. These data pave the way for Phase II studies in selected inherited and acquired liver disorders.
Asunto(s)
Hiperamonemia/terapia , Trasplante de Hígado , Hígado/metabolismo , Errores Innatos del Metabolismo/terapia , Trasplante de Células Madre , Edad de Inicio , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Amoníaco/metabolismo , Aciduria Argininosuccínica/metabolismo , Aciduria Argininosuccínica/patología , Aciduria Argininosuccínica/terapia , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hiperamonemia/metabolismo , Hiperamonemia/patología , Recién Nacido , Hígado/crecimiento & desarrollo , Hígado/patología , Masculino , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Células Madre/metabolismo , Urea/metabolismoRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). METHODS: Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. RESULTS: The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. CONCLUSIONS: We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.
Asunto(s)
Plaquetas/citología , Extractos Celulares , Células Madre Mesenquimatosas/citología , Animales , Bovinos , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Proliferación Celular/genética , Humanos , InmunofenotipificaciónRESUMEN
BACKGROUND: The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range. METHODS: For the endotoxin test we used a kinetic chromogenic LAL test. As this is a limit test for the control of impurities, in compliance with International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, we evaluated the specificity and detection limit.For the immunophenotype test, an identity test, we evaluated specificity through the Fluorescence Minus One method and we repeated all experiments thrice to verify precision. The immunophenotype validation required a performance qualification of the flow cytometer using two types of standard beads which have to be used daily to check cytometer reproducibly set up. The results were compared together.Collected data were statistically analyzed calculating mean, standard deviation and coefficient of variation percentage (CV%). RESULTS: The LAL test is repeatable and specific. The spike recovery value of each sample was between 0.25 EU/ml and 1 EU/ml with a CV% < 10%. The correlation coefficient (≥ 0.980) and CV% (< 10%) of the standard curve tested in duplicate showed the test's linearity and a minimum detectable concentration value of 0.005 EU/ml.The immunophenotype method performed thrice on our cell therapy products is specific and repeatable as showed by CV% inter -experiment < 10%. CONCLUSIONS: Our data demonstrated that validated analytical procedures are suitable as quality controls for the batch release of cell therapy products.Our paper could offer an important contribution for the scientific community in the field of CTPs, above all to small Cell Factories such as ours, where it is not always possible to have CFR21 compliant software.
Asunto(s)
Química Clínica/métodos , Química Clínica/normas , Control de Calidad , Animales , Anticuerpos/metabolismo , Células de la Médula Ósea/citología , Línea Celular Tumoral , Endotoxinas/metabolismo , Fluorescencia , Humanos , Inmunofenotipificación , Prueba de Limulus , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation. MSCs can express markers of the myogenic cell lineages and give rise, under specific cell culture conditions, to myotube-like structures. Nevertheless, we failed to obtain mixed myotubes both in vitro and in vivo. For in vivo transplantation, we tested a new protocol to collect human MSCs from whole bone marrow, to get larger numbers of cells. MSCs, when transplanted into the pelvic muscles close to the external urethral sphincter, survived for a long time in absence of immunosuppression, and migrated into the muscle among fibers, and towards neuromuscular endplates. Moreover, they showed low levels of cycling cells, and did not infiltrate blood vessels. We never observed formation of cell masses suggestive of tumorigenesis. Those which remained close to the injection site showed an immature phenotype, whereas those in the muscle had more elongated morphologies. Therefore, MSCs are safe and can be easily transplanted without risk of side effects in the pelvic muscles. Further studies are needed to elucidate their integration into muscle fibers, and to promote their muscular transdifferentiation either before or after transplantation.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Desarrollo de Músculos , Animales , Células de la Médula Ósea/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Técnicas de Cocultivo , Humanos , Inmunofenotipificación , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Mioblastos , Ratas , Incontinencia Urinaria/terapiaRESUMEN
Age-related macular degeneration (AMD) is a leading cause of legal blindness in individuals over 60 years of age, characterized by the dysfunction of retinal pigmented epithelium cells, specifically in the macular area. Despite several treatment options, AMD therapy remains difficult, especially for exudative AMD. Multipotent mesenchymal stem cells (MSCs), with great plasticity and immunomodulant properties, are a promising cell source for cellular therapy and tissue engineering. We evaluated the effects of steroid drugs, often used to treat AMD, in association with MSCs, in view of a possible application together to treat AMD. Morphology, viability, growth kinetics, and immunophenotype were evaluated on healthy donors' MSCs, treated with triamcinolone acetonide, alcohol-free triamcinolone acetonide, micronized intravitreal triamcinolone and dexamethasone at different concentrations, and in a human retinal pigment epithelial cell line supernatant (ARPE-19). The morphological analysis of MSCs in their standard medium showed a negative correlation with drug concentrations, due to the numerous crystals. Dexamethasone was the least toxic corticosteroid used in this study. ARPE-19 seemed to help cells preserve the typical MSC morphology. In conclusion, this in vitro study demonstrated that high doses of corticosteroid drugs have a negative effect on MSCs, reduced in the presence of a conditioned media.
RESUMEN
BACKGROUND: The quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests' accuracy, precision, repeatability, linearity and range. METHODS: As the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator. RESULTS: All the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128. CONCLUSIONS: Our data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution).
Asunto(s)
Laboratorios , Control de Calidad , Humanos , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
The main limit of umbilical cord blood hematopoietic stem cell transplantation is a more difficult engraftment related to the number of cells infused per kilogram of recipient body weight. This limit makes the cord blood a suboptimal source of hematopoietic stem cells for transplantation in case of difficult engraftment situations. Direct intrabone cord blood (CB) injection has been recently investigated as a solution to cell dose problem in the adults population, but there is a lack of data concerning this approach in pediatric patients. Here, we describe 5 pediatric patients undergoing intrabone cord blood transplantation (IBCBT) for different diseases characterized by a high risk of posttransplant graft failure. The conditioning regimen differed according to the disease, whereas the GvHD prophylaxis consisted of cyclosporine, mycophenolate, and ATG. The median numbers of total nucleated cells infused and CD34(+) cells were 3.3 × 10(7)/kg, 2 × 10(5)/kg. All the patients showed complete hematological recovery and complete donor engraftment. No patient had secondary graft failure, whereas 1 patient relapsed 6 months after IBCBT. No patient died of transplant-related complications. Our results show that IBCBT is safe and feasible in pediatrics as well, and suggest that IBCBT might be an attractive option to overcome some limits of umbilical cord blood hematopoietic stem cell transplantation.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Adolescente , Niño , Preescolar , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Lactante , Masculino , Proyectos Piloto , Riesgo , Acondicionamiento PretrasplanteRESUMEN
Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of "optimal" conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm(2). After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm(2). Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm(2) did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use.
RESUMEN
BACKGROUND AIMS: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS: The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS: Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS: Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.
Asunto(s)
Vasos Coronarios/cirugía , Ligadura , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/terapia , Animales , Antígenos CD/metabolismo , Antígenos CD34/biosíntesis , Médula Ósea/patología , Cadherinas/metabolismo , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Endotelio/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos NOD , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Recuperación de la FunciónRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective therapies. Cell replacement therapy has been suggested as a promising therapeutic approach for multiple neurodegenerative diseases, including motor neuron disease. We analyzed expanded mesenchymal stem cells (MSCs) isolated from sporadic ALS patients and compared them with MSCs isolated from healthy donors. MSCs were isolated from bone marrow by Percoll gradient and maintained in culture in MSC Medium until the third passage. Growth kinetics, immunophenotype, telomere length, and karyotype were evaluated during in vitro expansion. Osteogenic, adipogenic, chondrogenic, and neurogenic differentiation potential were also evaluated. No morphological differences were observed in the MSCs isolated from donors or patients. The cellular expansion potential of MSCs from donors and patients was slightly different. After three passages, the MSCs isolated from donors reached a cumulative population doubling higher than from patients but the difference was not statistically significant. No significant differences between donors or patients were observed in the immunophenotype analysis. No chromosomal alteration or evidence of cellular senescence was observed in any samples. Both donor and patient MSCs, after exposure to specific conditioning media, differentiated into adipocytes, osteoblasts, chondrocytes, and neuron-like cells. These results suggest that extensive in vitro expansion of patient MSCs does not involve any functional modification of the cells, including chromosomal alterations or cellular senescence. Hence, there is a good chance that MSCs might be used as a cell-based therapy for ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Adulto , Anciano , Esclerosis Amiotrófica Lateral/sangre , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Proliferación Celular , Femenino , Citometría de Flujo , Humanos , Cariotipificación , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Telómero/genéticaRESUMEN
As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Antígenos CD34/análisis , Terapia Genética/métodos , VIH-1/genética , Animales , Células Sanguíneas/fisiología , Células de la Médula Ósea/fisiología , Citometría de Flujo , Genes Reporteros , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Proteínas Fluorescentes Verdes/genética , Movilización de Célula Madre Hematopoyética , Humanos , Inmunofenotipificación , Interleucina-3/farmacología , Ratones , Ratones SCID , Proteínas Recombinantes/farmacología , Trombopoyetina/farmacologíaRESUMEN
OBJECTIVE: Several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (HSCs). Because most cord blood (CB) samples are frozen in single bags and only an aliquot ( approximately 25%) of the blood can be expanded, the thawing and refreezing of samples must be validated in the current European and Italian Good Manufacturing Practice (eIGMP) conditions. Here, we describe in vitro and in vivo validation of the phase I/II protocol for CD34+ expansion of thawed CB units according to the current Cell Therapy Products (CTPs) Guidelines. MATERIALS AND METHODS: CB units were thawed and 25% of the total volume was processed for CD34+ selection by CliniMACS. The 75% of the unit was immediately refrozen. CD34+ cells were expanded for 3 weeks with stem cell factor, Flt-3/Flk-2 ligand, thrombopoietin, and interleukin-6. RESULTS: In vitro results demonstrated that this culture system induces expansion of thawed CD34+ (median value = 8.3). In vivo data demonstrated that after culture, the final CTPs maintain their repopulating ability in nonobese diabetic severe combined immunodeficient (SCID) mice. Limiting dilution assays performed by injecting decreasing doses of expanded CD34+ cells revealed that the frequency of SCID repopulating cells after ex vivo expansion is 1:8,034. Analyses for sterility, viability, cell senescence, and cytogenetic assessment demonstrated that expansion procedures in eIGMP conditions are safe for clinical protocols. CONCLUSIONS: This offers promising new options for expansion of allogenic HSCs and also for autologous usage in transplantation and other cell therapy protocols.
Asunto(s)
Proliferación Celular , Trasplante de Células Madre de Sangre del Cordón Umbilical , Criopreservación , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Animales , Antígenos CD34 , Separación Celular , Guías como Asunto , Células Madre Hematopoyéticas/fisiología , Humanos , Italia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante Heterólogo , Trasplante HomólogoRESUMEN
Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated. For a possible clinical application, all experiments were carried out in serum-free media. A low-affinity nerve growth factor receptor (LNGFR) pseudotyped murine retroviral vector was used to transduce cord blood CD34(+) cells, which were then expanded ex vivo. These cells engrafted up to three generations of serially transplanted nonobese diabetic/severe combined immunodeficiency mice: 54.26% +/- 5.59%, 19.05% +/- 2.01%, and 6.15% +/- 5.16% CD45(+) cells from primary, secondary, and tertiary recipient bone marrow, respectively, were LNGFR(+). Repopulation in secondary and tertiary recipients indicates stability of transgene expression and long-term self-renewal potential of transduced HSCs, suggesting that retroviral gene transfer into HSCs, followed by ex vivo expansion, could facilitate long-term engraftment of genetically modified HSCs.
Asunto(s)
Antígenos CD34/biosíntesis , Proliferación Celular , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Retroviridae/genética , Inmunodeficiencia Combinada Grave/terapia , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Sangre Fetal/inmunología , Sangre Fetal/trasplante , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inmunodeficiencia Combinada Grave/genética , Transgenes , Trasplante HeterólogoRESUMEN
Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti-ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both--such as trastuzumab or gefitinib--might increase the efficiency of purging.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Células Madre Hematopoyéticas/citología , Separación Inmunomagnética , Proteínas Oncogénicas v-erbB/antagonistas & inhibidores , Trasplante de Células Madre de Sangre Periférica/métodos , Adulto , Antígenos CD34 , Técnicas de Cultivo de Célula/métodos , Femenino , Humanos , Persona de Mediana Edad , Células Neoplásicas Circulantes/patología , Proteínas Oncogénicas v-erbB/inmunología , Trasplante Autólogo , Carga TumoralRESUMEN
We have previously established a stroma-free culture with Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long-lasting hematopoietic repopulation in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. In this work the kinetics of megakarocyte (Mk)-engraftment that is often poor and delayed in CB transplantation, and human platelet (HuPlt) generation in NOD/SCID mice of baseline CD34+ cells (b34+), and of CD34+ cells reisolated after a 4-week expansion with FL+SCF+TPO (4w34+) were compared. With b34+ cells Mk-engraftment was first seen at week 3 (CD41+: 0.4%); 4w34+ cells allowed a more rapid Mk-engraftment (at weeks 2 and 3 the CD41+ cells were 0.3% and 0.8%). Circulating HuPlts were first seen at weeks 2 and 1, respectively. Mk-engraftment levels of b34+ and 4w34+ cells 6-8 weeks after transplantation were similar (12 +/- 3.5 versus 15 +/- 5% CD45+; 1.3 +/- 0.5 versus 1.8 +/- 0.5% CD41+ cells). Also serial transplant experiments were performed with expanded and reselected CB cells. In secondary and tertiary recipients the Mk population was detected with bone marrow fluorescence-activated cell sorter analysis; these experiments indicate the effective long-term repopulation of expanded cells. Selected CD34+ cells after a 4-week expansion with FL+SCF+TPO are more efficient in Mk engraftment than the same number of unmanipulated cells.
Asunto(s)
Sangre Fetal/trasplante , Trasplante de Células Madre Hematopoyéticas/métodos , Megacariocitos/trasplante , Transfusión de Plaquetas , Animales , Antígenos CD34/inmunología , Médula Ósea/inmunología , Sangre Fetal/inmunología , Humanos , Megacariocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Telomerase activity, telomere length, stem/progenitor cell production, and function of CD34+ cells from cord blood (CB), bone marrow, and mobilized peripheral blood were evaluated in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34+ cells reisolated at monthly intervals for passage. Continuous expansion of stem cells as measured by in vitro cobblestone area and secondary colony-forming assays was noted for 18 to 20 weeks and by severe combined immunodeficiency (SCID)-repopulating cells (SRCs), capable of repopulating and serially passage in nonobese diabetic/SCID mice, for 16 weeks. Despite this extensive proliferation, telomere length initially increased and only at late stages of culture was evidence of telomere shortening noted. This telomere stabilization correlated with maintenance of high levels of telomerase activity in the CD34+ cell population for prolonged periods of culture. Cytokine-stimulated cultures of adult CD34+ cells showed CD34+ and SRC expansion (6-fold) for only 3 to 4 weeks with telomere shortening and low levels of telomerase. There is clearly a clinical value for a system that provides extensive stem cell expansion without concomitant telomere erosion.
Asunto(s)
Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Telomerasa/sangre , Telomerasa/metabolismo , Adenoviridae/genética , Animales , Antígenos CD/sangre , Antígenos CD34/sangre , División Celular , Vectores Genéticos , Humanos , Recién Nacido , Ratones , Ratones Endogámicos NOD , Ratones SCID , Telomerasa/deficiencia , Trombopoyetina/genética , TransfecciónRESUMEN
BACKGROUND AND OBJECTIVES: Megakaryocyte (Mk) engraftment is often poor and delayed after cord blood (CB) transplantation. Ex vivo manipulations of the cells that will be infused may be a way to achieve better Mk engraftment. In this study we investigated the ability of different hematopoietic growth factor combinations to generate large numbers of Mk cells ex vivo. DESIGN AND METHODS: To find the best cytokine combination capable of generating large numbers of Mks, baseline CB CD34+ (bCD34+) cells and CD34+ and CD34- cells, immunoselected after 4 weeks of expansion with thrombopoietin (TPO), stem cell factor (SCF) and Flt-3 ligand (FL) (eCD34+, eCD34-), were further cultured in the presence of different cytokine combinations (containing interleukin(IL)-3, SCF, TPO and IL-6). To evaluate Mk reconstitution in vivo, Mk-committed cells, generated during 10 days of in vitro culture, were injected into NOD/SCID mice and the kinetics of human platelet production was evaluated. RESULTS: TPO and SCF together were found to be sufficient to generate large numbers of Mk cells (3 +/- 0.40 x 10(6)/1 x 10(5) input bCD34+ cells) from bCD34+ cells; the addition of IL-3 and IL-6 did not further increase Mk production (3.5 +/- 0.63 x 10(6)/1 x 10(5) input bCD34+ cells). In contrast only one cytokine combination (IL-3+SCF+IL-6+TPO) induced a large Mk production from eCD34+ and eCD34- cells (0.16 +/- 0.04 x 10(6)/1 x 10(5) input eCD34+ cells and 0.035 x 10(6) +/- 0.012 x 106/1 x 10(5) input eCD34- cells, respectively). In mice injected with Mk-committed cells derived from bCD34+ or eCD34+ cells, human platelets were first detected on day 3 and disappeared after 4 weeks; in mice injected with MK-committed cells derived from eCD34- cells, human platelets peaked at day 3, but disappeared quickly. INTERPRETATION AND CONCLUSIONS: Fast Mk-engraftment can be obtained by in vitro selective lineage-commitment of baseline and ex vivo expanded CB cells.
Asunto(s)
Sangre Fetal/citología , Megacariocitos/citología , Megacariocitos/trasplante , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Trasplante de Células , Citocinas/farmacología , Supervivencia de Injerto , Humanos , Ratones , Ratones SCID , Trasplante HeterólogoRESUMEN
OBJECTIVE: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. METHODS: Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. RESULTS: Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity. CONCLUSION: We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.