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Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications.
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Células Madre Mesenquimatosas/inmunología , Linfocitos T/inmunología , Líquido Amniótico/citología , Células de la Médula Ósea/citología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Placenta/citología , Embarazo , Subgrupos de Linfocitos T , Linfocitos T/citologíaRESUMEN
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.
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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.
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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
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).
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Laboratorios , Control de Calidad , Humanos , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
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.
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Mobilization of endothelial progenitor cells has been suggested to contribute to neo-vascularization of ischemic organs. Aim of this study was to investigate whether the combination of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF may influence the expansion of circulating KDR+ cells in patients with acute myocardial infarction (AMI). KDR+ cells significantly increased in peripheral blood of AMI patients treated with G-CSF and GM-CSF compared to untreated patients. This KDR+ cells population was CD14+ but not CD34+ or CD133+. CD14+/KDR+ cells were also obtained in vitro by culturing mononuclear cells from healthy donors in a Rotary Cell Culture System in the presence of G-CSF + GM-CSF, but not of the individual growth factors. CD14+/KDR+ cells, obtained from patients or from in vitro culture, co-expressed hematopoietic (CD45, CD14) and endothelial markers (CD31, CD105, and VE-cadherin). CD14+/KDR+, but not CD14+/KDR- cells, stimulated the organization of human microvascular endothelial cells into capillary-like structures on Matrigel both in vitro and in vivo. The combination of G-CSF and GM-CSF induced a CD14+/KDR+ cell population with potential pro-angiogenic properties.
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Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Monocitos/metabolismo , Infarto del Miocardio/metabolismo , Neovascularización Patológica , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Antígenos CD34/biosíntesis , Células Cultivadas , Células Endoteliales/citología , Glicoproteínas/biosíntesis , Humanos , Técnicas In Vitro , Receptores de Lipopolisacáridos/biosíntesis , Ratones , Ratones SCID , Péptidos , Células Madre/citología , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: To evaluate feasibility, safety and pattern of bone marrow-derived cells (BMC) mobilization in patients with end stage liver cirrhosis following granulocyte-colony stimulating factor (G-CSF) administration. METHODS: Eight patients with severe liver cirrhosis (Child-Pugh score B-C, spleen diameter less than 170 mm) were included. They were treated with G-CSF (5 microg/kg b.i.d for three consecutive days) to mobilize BMC, evaluated as circulating CD34+ve cells (flow cytometry) and myeloid CFU-GM progenitors (in vitro colony growth assay). Co-expression in CD34+ve cells markers of differentiation (Thy1, CD133, CXCR4, c1qRp) were investigated on CD34+ve cells by double direct immunofluorescence. Data from 40 healthy haematopoietic stem cell donors were used as controls. RESULTS: Mobilization of CD34+ve cells occurred in all patients. It was paralleled by expansion of circulating CFU-GM progenitors. Circulating CD34+ve cells co-expressed epithelial and stem cell markers in both cirrhotics and volunteer stem cell donors. G-CSF was well tolerated, no adverse event occurred, a significant reversible increase of splenic longitudinal diameter was observed. CONCLUSIONS: (i) G-CSF mobilization of BMC co-expressing epithelial and stem markers occurred in all cirrhotic patients; (ii) splenomegaly up to 170 mm does not prevent safe BMC mobilization following G-CSF in patients with end stage liver disease; (iii) mobilized BMC may represent an easy immature cell source potentially useful for novel approaches for liver regeneration.
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Células de la Médula Ósea/fisiología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Movilización de Célula Madre Hematopoyética/métodos , Encefalopatía Hepática/terapia , Anciano , Antígenos CD/sangre , Antígenos CD34/sangre , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Femenino , Factor Estimulante de Colonias de Granulocitos/toxicidad , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Bazo/patología , Trasplante de Células Madre/efectos adversos , Células Madre/citología , Células Madre/efectos de los fármacos , Resultado del TratamientoRESUMEN
Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment. Since CB units have, to date, mainly been stored in individual bags, our results suggest new options for optimizing the timing of infusions of expanded and non-expanded progenitors in transplants.
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Criopreservación/métodos , Sangre Fetal/trasplante , Trasplante de Células Madre Hematopoyéticas/métodos , Animales , Células Cultivadas , Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Especificidad de la EspecieRESUMEN
AIMS: To verify feasibility and safety of bone marrow stem cells (BMC) mobilization in patients (pts) with acute myocardial infarction (AMI) and to monitor the clinical effects of BMC mobilization in terms of myocardial perfusion and function. METHODS AND RESULTS: Eight male pts (median age: 50.5 years) treated with a primary PTCA were enrolled. The mobilization regimen consisted of G-CSF 5 microg/kg/12 h from day 0 to day +2 and GM-CSF 2.5 microg/kg/24 h. All pts underwent coronary angiography, intracoronary doppler flow study, echocardiography, and nuclear thallium scan before treatment and at 6 months. All pts showed increased values of WBC and circulating CD34+ following cytokine administration. No patient died. All patients completed a 6-months follow-up: target lesion revascularization rate was 12,5%, target vessel revascularization rate was 37.5%, angiographic mean ejection fraction increased from 49.8+/-11.9 to 55.4+/-8.7 (p=NS), mean coronary flow reserve from 1.63+/-0.42 to 2.5+/-0.4 (p=0.001), mean Thallium uptake raised from 55.56+/-16.42% to 67.56+/-13.66% (p=0.01), and normally perfused segments from 16% to 52% (p=0.01). CONCLUSION: Cytokine-induced BMC mobilization is feasible in AMI pts. Improvements of myocardial perfusion can be expected after PTCA associated with G-CSF and GM-CSF induced mobilization. Further studies are required to define the role of BMC-mobilization and the most effective cytokine combination.
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OBJECTIVE: Cord blood CD34+ cells are more uncommitted than their adult counterparts as they can be more easily maintained and expanded in vitro and transduced with lentiviral vectors. The aim of this study was to evaluate whether pretreatment with high-energy shock waves (HESW) could further enhance the expansion of cord blood progenitors and the transduction efficiency with lentiviral vectors. METHODS: Human cord blood CD34+ cells underwent HESW treatment with a wide range of energy and number of shots (from 0.22 mJ/mm2 to 0.43 mJ/mm2 and from 200 to 1500 shots). Cells were then evaluated both for their in vitro expansion ability and in vivo engraftment in primary, secondary, and tertiary NOD/SCID mice. The transduction efficiency with a lentiviral vector (LV) was also evaluated in vitro and in vivo. RESULTS: Cell viability following HESW ranged from 75 to 92%. Pretreatment with HESW significantly improved early progenitor cell expansion after short-term suspension culture. Upon transplantation in primary NOD/SCID mice, the HESW treatment enhanced progenitor cell engraftment (total human CD45(+)CD34+ cells were 10% in controls and 14.5% following HESW, human CD45(+)CD34(+)CD38(-) cells were 0.87% in controls and 1.8% following HESW). HESW treatment enhanced the transduction of a GFP+ lentiviral vector (e.g., at day 42 of culture 6.5% GFP+ cells in LV-treated cell cultures compared to 11.4% of GFP+ cells in HESW-treated cell cultures). The percentage of human GFP+ cell engrafting NOD/SCID mice was similar (34% vs 26.4% in controls); however, the total number of human cells engrafted after HESW was higher (39.6% vs 15%). CONCLUSION: The pretreatment of CD34+ cells with HESW represents a new method to manipulate the CD34+ population without interfering with their ability to both expand and engraft and it might be considered as a tool for genetic approaches.
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Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de la radiación , Ondas de Choque de Alta Energía , Animales , Antígenos CD34 , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Estudios de Factibilidad , Supervivencia de Injerto/efectos de la radiación , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante HeterólogoRESUMEN
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.
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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
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.
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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.
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Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Animales , Células Sanguíneas , Células de la Médula Ósea , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Citocinas/farmacología , Supervivencia de Injerto , Hematopoyesis , Movilización de Célula Madre Hematopoyética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante HeterólogoRESUMEN
The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 x 10(5) CD34(+) cells, the percentages of CD45(+) cells were 20.5 +/- 4.5 [week 1, expanded] and 27.2 +/- 8.2 [week 4, expanded] vs 11.7 +/- 2.5 [nonexpanded]; n = 5). The GFP(+)/CD45(+) cell fraction was similar in all cases (12.5% +/- 2.9% and 12.2% +/- 2.7% vs 12.7% +/- 2.1%). Engraftment was multilineage, with GFP(+)/lineage(+) cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% +/- 4.3% (n = 12); 19.7% +/- 6.2% of human cells were GFP(+). In tertiary transplants the percentage of CD45(+) cells was lower (4.3% +/- 1.7%; n = 10); 14.8% +/- 5.9% of human cells were GFP(+), and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.