RESUMO
To determine the therapeutic efficacy of human umbilical cord lining mesenchymal stromal cells (CL-MSCs) (US Patent number 9,737,568) in lupus-prone MRL/lpr (Faslpr) mice and elucidate its working mechanisms. A total of 4 doses of (20-25) × 106 cells/kg of CL-MSCs was given to 16-week-old female Faslpr mice by intraperitoneal injection. Three subsequent doses were given on 17 weeks, 18 weeks, and 22 weeks, respectively. Six-week-old Faslpr mice were used as disease pre-onset controls. Mice were monitored for 10 weeks. Mouse kidney function was evaluated by examining complement component 3 (C3) deposition, urinary albumin-to-creatinine ratio (ACR), and lupus nephritis (LN) activity and chronicity. Working mechanisms were elucidated by flow cytometry, Luminex/ELISA (detection of anti-dsDNA and isotype antibodies), and RNA sequencing. CL-MSCs improved mice survival and kidney function by reducing LN activity and chronicity and lymphocyte infiltration over 10 weeks. CL-MSCs also reduced urinary ACR, renal complement C3 deposition, anti-dsDNA, and isotype antibodies that include IgA, IgG1, IgG2a, IgG2b, and IgM. Immune and cytokine profiling demonstrated that CL-MSCs dampened inflammation by suppressing splenic neutrophils and monocytes/macrophages, reducing plasma IL-6, IL-12, and CXCL1 and stabilizing plasma interferon-γ and TNF-α. RNA sequencing further showed that CL-MSCs mediated immunomodulation via concerted action of pro-proinflammatory cytokine-induced chemokines and production of nitric oxide in macrophages. CL-MSCs may provide a novel myeloid (neutrophils and monocytes/macrophages)-targeting therapy for SLE.
Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Células-Tronco Mesenquimais , Feminino , Humanos , Animais , Camundongos , Camundongos Endogâmicos MRL lpr , Rim/metabolismo , Citocinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/metabolismo , Lúpus Eritematoso Sistêmico/terapiaRESUMO
The immunosuppressive properties of mesenchymal stromal cells (MSCs) have been clinically proven to be effective in treating graft-versus-host disease (GVHD). However, MSC therapy is limited by the need for laborious and expensive manufacturing processes that are fraught with batch-to-batch variability. Substitution of MSC therapy with key MSC-mediated immunomodulatory factors could be an option for GVHD treatment. Using a simulated in vitro model of the immunosuppressive effects of MSC on allogeneic graft reactions, a synergistic 2-factor combination (2FC) of CXCL5 and anti-CCL24 was identified from a panel of over 100 immunomodulatory factors as being superior to MSCs in the modulation of mixed lymphocyte reactions. This 2FC was superior to cyclosporine in ameliorating both moderate and severe GVHD while being equivalent to MSCs in moderate GVHD and superior to MSCs in severe GVHD. Its immunosuppressive efficacy could be further improved by extended treatment. Mechanistic studies revealed that in vitro the 2FC could only reduce the proliferation of Th 1 and Th 17, whereas in vivo CXCL5 acts in concert with anti-CCL24 antibody to reduce not only transplanted Th 1 and Th 17 but also cytotoxic T lymphocytes and natural killer cells to increase mouse immunosuppressive neutrophils without affecting human hematopoietic stem cell reconstitution. Concurrently, it reduced circulating human proinflammatory cytokines IFN-γ, IL-6, IL-17A, IL-8, macrophage inflammatory protein-1ß, and monocyte chemoattractant protein-1. Both in vitro and in vivo data suggest that CXCL5 and anti-CCL24 antibody act in concert to ameliorate GVHD via suppression of Th 1 and Th 17 responses. We propose that this novel 2FC could substitute for MSC therapy in GVHD treatment.
Assuntos
Quimiocina CCL24/farmacologia , Quimiocina CXCL5/farmacologia , Ciclosporina/farmacologia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Xenoenxertos , Humanos , Linfócitos/imunologia , Linfócitos/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Telômero/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Engenharia Genética , Genoma Humano , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/transplante , Humanos , Camundongos SCID , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transplante de Células-Tronco , Telomerase/metabolismo , Encurtamento do Telômero/efeitos dos fármacos , Teratoma/genética , Teratoma/patologiaRESUMO
The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.
Assuntos
Células-Tronco Hematopoéticas/imunologia , Células-Tronco Mesenquimais/imunologia , Nicho de Células-Tronco/imunologia , Reatores Biológicos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaios Clínicos como Assunto , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Citocinas/farmacologia , Etilenodiaminas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Proteínas de Membrana/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Niacinamida/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/imunologiaRESUMO
In this study, we report that surface functional groups of single walled carbon nanotubes (f-SWCNT) are critical for mediating survival and ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood (UCB). In comparison to amide (-O-NH2) and polyethylene-glycol (-PEG) functionalized SWCNT, carboxylic acid (-COOH) variants gave optimal viability support which correlated with maximal reduction of lethal mitochondrial superoxides in HSPC. Cytokine array illustrated that f-SWCNT-COOH maintained higher proportion of HSPC associated cytokines and minimal level of differentiation promoting factors. Transplantation of f-SWCNT-COOH expanded grafts in sub-lethally irradiated immunodeficient mice resulted in higher engraftment of HSPC in bone marrow compared to control when they were co-transplanted with non-expanded cells from the same UCB. Expanded grafts mediated higher survival rate of mice compared to non-expanded grafts due to lower graft-versus-host-disease which is likely a consequence of proportion of immune cells in the grafts. FROM THE CLINICAL EDITOR: Umbilical cord blood (UCB) is a potential source of hematopoietic stem and progenitor (HSPC) cells. One major hurdle for its clinical use is the insufficient yield of cell number. The authors in this study elegantly demonstrated the importance of various functional groups on single-walled carbon nanotubes (f-SWCNT) in enhancing ex vivo expansion of HSPC in UCB. The findings may pave a way for having UCB as a source for HSPC for clinical use in the future.
Assuntos
Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Nanotubos de Carbono/química , Animais , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Sangue Fetal/efeitos dos fármacos , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanotubos de Carbono/efeitos adversos , Superóxidos/metabolismo , Transplante HeterólogoRESUMO
BACKGROUND AIMS: Double cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment. METHODS: Post-thaw cryopreserved CBUs from cord blood banks, hereinafter termed "banked" CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days. RESULTS: When DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma(null) (NOD/SCID-IL2γ(-/-), NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0-66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0-92.9%) for mice receiving an expanded unit (P < 0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture. CONCLUSIONS: MSC-supported ex vivo expansion of "banked" CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.
Assuntos
Células da Medula Óssea/citologia , Sangue Fetal/transplante , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Mesenquimais/citologia , Linfócitos T Reguladores/citologia , Animais , Células Cultivadas , Técnicas de Cocultura , Sangue Fetal/citologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Hematopoese , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Camundongos , Transplante HeterólogoRESUMO
In this study, carboxylic acid functionalized single walled carbon nanotubes (f-SWCNT-COOH) was shown to support the viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood-mononucleated cells (UCB-MNC). Our in vitro experiments showed that f-SWCNT-COOH increased the viability of the CD45(+) cells even without cytokine stimulation. It also reduced mitochondrial superoxides and caspase activity in CD45(+) cells. f-SWCNT-COOH drastically reduced the proportions of CD45(-) cells in the non-enriched UCB-MNC. Phenotypic expression analysis and functional colony forming units (CFU) showed significant ex vivo expansion of HSPC, particularly of CD45(+)CD34(+)CD38(-) population and granulocyte-macrophage (GM) colonies, in f-SWCNT-COOH augmented cultures supplemented with basal cytokines. In vivo data suggested that f-SWCNT-COOH expanded UCB-MNC could repopulate immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease (GVHD) and f-SWCNT-COOH dependent toxicity. FROM THE CLINICAL EDITOR: In this paper a novel method is presented by using single wall functionalized carbon nanotubes to enhance viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells in human umbilical cord blood -mononucleated cells. Detailed data is presented about enhanced viability, including improved repopulation of immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease.
Assuntos
Sangue Fetal/citologia , Sangue Fetal/transplante , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Nanotubos de Carbono/química , ADP-Ribosil Ciclase 1/análise , Animais , Antígenos CD34/análise , Ácidos Carboxílicos/química , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Congelamento , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Antígenos Comuns de Leucócito/análise , Camundongos , Camundongos SCIDRESUMO
BACKGROUND: Studies have reported the use of repetitive transcranial magnetic stimulation (rTMS) in patients with major depressive disorder (MDD). However, most studies focus on antidepressant effect of rTMS, but few on cognitive aspects. The present study aimed to explore the effect of rTMS on BDNF levels and cognitive function in the treatment of middle-aged and elderly MDD. METHODS: This was a randomized controlled trial. A total of 120 elderly patients with MDD treated in The Second Affiliated Hospital of Xi'an Medical University from January 2021 to January 2023 were selected as research subjects. The patients were randomly divided into control group (nâ =â 60, patients received simple oral treatment with escitalopram and sham rTMS) and study group (nâ =â 60, patients received oral treatment with escitalopram combined with rTMS) according to the random number table method. We compared the clinical efficacy, serum BDNF levels, and cognitive function between the 2 groups. RESULTS: After treatment, the HAMD-17 score in the study group was lower than that in the control group [13.00 (12.00-16.00) vs 17.00 (15.00-19.00), Pâ <â .05], and the RBANS score was higher than that in the control group [166.00 (161.25-171.75) vs 133.00 (130.00-136.75), Pâ <â .05]. The total effective rate of the research group was 95.0%, which was higher than the 82.0% of the control group (Pâ <â .05). The serum BDNF levels [36.00 (33.00-38.00) vs 30.00 (28.00-32.00), Pâ <â .05] and MoCA scores [24.00 (22.00-26.75) vs 23.00 (21.00-25.00), Pâ <â .05] of the study group were higher than those of the control group. There were no significant adverse reactions during the treatment of both groups. CONCLUSIONS: Compared with oral escitalopram alone, repeated transcranial magnetic stimulation in the treatment of middle-aged and elderly patients with major depressive disorder can further improve the efficacy, and can more effectively improve the BDNF level and cognitive function, with ideal safety.
Assuntos
Transtorno Depressivo Maior , Idoso , Pessoa de Meia-Idade , Humanos , Transtorno Depressivo Maior/terapia , Estimulação Magnética Transcraniana , Escitalopram , Fator Neurotrófico Derivado do Encéfalo , Administração OralRESUMO
Human cord blood-derived γδ T cells (CBγδ) display a highly diverse TCRγδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCRγδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2- clones compared to Vδ2+ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.
Assuntos
Infecções por Vírus Epstein-Barr , Linfócitos T , Adulto , Humanos , Sangue Fetal , Herpesvirus Humano 4 , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
OBJECTIVE: To determine the efficacy of CXCL5 administration in lupus-prone MRL/lpr (Faslpr ) mice and elucidate its working mechanisms. METHODS: CXCL5 expression in blood (obtained from SLE patients and Faslpr mice) and major internal organs (obtained from Faslpr mice) was examined by Luminex, real-time polymerase chain reaction, and immunofluorescent staining analyses. Pharmacokinetic studies were performed in Faslpr mice and healthy Institute of Cancer Research mice. Efficacy of CXCL5 administration was demonstrated in Faslpr mice, and the working mechanism of CXCL5 treatment was elucidated by flow cytometry, Luminex, and RNA sequencing. RESULTS: In SLE patients, serum CXCL5 levels were significantly lower than in healthy individuals (P < 0.0001) and negatively correlated with disease activity (P = 0.004). In Faslpr mice, disease severity progressed with age and was negatively associated with plasma CXCL5 levels. Intravenous administration of CXCL5 to Faslpr mice restored endogenous circulatory CXCL5, improved mice survival, and reduced anti-double-stranded DNA antibodies, proteinuria, lupus nephritis activity and chronicity indices, renal complements, and neutrophil extracellular traps over short-term (10 weeks) and long-term (2 years) time periods. In vitro and in vivo assays demonstrated that CXCL5 dictated neutrophil trafficking and suppressed neutrophil activation, degranulation, proliferation, and renal infiltration. Renal and splenic RNA sequencing further showed that CXCL5-mediated immunomodulation occurred by promoting energy production in renal-infiltrated immune cells, activating certain T cells, and reducing tissue fibrosis, granulocyte extravasation, complement components, and interferons. Further factorial design results indicated that CXCL5 appears to enhance host tolerability to cyclophosphamide in vulnerable individuals. CONCLUSION: We found that serum CXCL5 levels were significantly lower in SLE patients than in healthy individuals and were negatively correlated with disease activity. By administering CXCL5 intravenously in a mouse model of lupus, mouse survival improved, and indices of disease activity reduced significantly. Taken together, these findings indicate CXCL5 administration may represent a novel myeloid/neutrophil-targeting therapy for SLE.
Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Camundongos , Animais , Neutrófilos/metabolismo , Camundongos Endogâmicos MRL lpr , Rim/metabolismo , Inflamação/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismoRESUMO
Ex vivo expansion of cord blood (CB) hematopoietic stem cells and cotransplantation of 2 CB units (CBUs) could enhance the applicability of CB transplantation in adult patients. We report an immunodeficient mouse model for cotransplantation of ex vivo expanded and unexpanded human CB, showing enhanced CB engraftment and provide proof of concept for this transplantation strategy as a means of overcoming the limiting cell numbers in each CBU. CBUs were expanded in serum-free medium supplemented with stem cell factor, Flt-3 ligand, thrombopoietin, and insulin growth factor binding protein-2 together with mesenchymal stromal cell coculture. Unexpanded and expanded CB cells were cotransplanted by tail vein injection into 45 sublethally irradiated nonobese diabetic SCID-IL2γ(-/-) (NSG) mice. Submandibular bleeding was performed monthly, and mice were sacrificed 4 months after transplantation to analyze for human hematopoietic engraftment. Expansion of non-CD34(+) selected CB cells yielded 40-fold expansion of CD34(+) cells and 3.1-fold expansion of hematopoietic stem cells based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts exhibited 4.30% human cell repopulation, compared with 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis (P = .07). Ex vivo expanded grafts with lower initiating cell doses also showed equivalent engraftment to unexpanded grafts with higher cell dose (8.0% versus 7.9%; P = .93). In conclusion, ex vivo expansion resulted in enhanced CB engraftment despite eventual rejection by the unexpanded CBU.
Assuntos
Sangue Fetal/transplante , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Antígenos CD34/biossíntese , Antígenos CD34/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Sangue Fetal/citologia , Sangue Fetal/imunologia , Sobrevivência de Enxerto/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Injeções Intravenosas , Proteínas de Membrana/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos SCID , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , Transplante Heterólogo , Irradiação Corporal TotalRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. METHODS: In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). RESULTS: Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P < 0.01). This was associated with significant enhancement of mitochondrial membrane potential (P < 0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P < 0.0001). CONCLUSIONS: Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.
Assuntos
Técnicas de Cultura de Células , Citosol/metabolismo , Sangue Fetal/citologia , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Animais , Morte Celular/genética , Fusão Celular , Proliferação de Células , Sobrevivência Celular , Transdiferenciação Celular , Técnicas de Cocultura , Proteínas de Fluorescência Verde/análise , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Células NIH 3T3RESUMO
The expansion of umbilical cord blood mononuclear cells (UCB MNCs) was investigated in a novel co-culture system by means of encapsulation of rabbit bone marrow (BM) mesenchymal stem cells (MSCs) in alginate beads (Alg beads). Three kinds of media were applied and the experiments lasted for 7 days. The total nucleated cell density was measured every 24 h. Flow cytometric assay for CD34(+) cells and methylcellulose colony assays were carried out at 0, 72 and 168 h. It was found that the encapsulated MSCs illustrated remarkable effects on UCB MNCs expansion regardless of whether serum is present in culture media or not. At the end of 168 h co-culture, the total nucleated cell number was multiplied by 15 +/- 2.9 times, and CD34(+) cells 5.3 +/- 0.3 times and colony-forming units in culture (CFU-Cs) 5.6 +/- 1.2 times in the serum-free media supplemented with conventional dose of cytokines, which was very similar to the results in the containing 20% serum media. While in the control, i.e. MNC expansion without encapsulated MSCs, however, total nucleated cells density changed mildly, CD34(+) cells and CFU-Cs showed little effective expansion. It is demonstrated that the encapsulated stromal cells can support the expansion of UCB MNCs effectively under the experimental condition.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Veias Umbilicais/citologia , Alginatos/química , Animais , Antígenos CD34/biossíntese , Diferenciação Celular , Técnicas de Cocultura , Composição de Medicamentos , Ácido Glucurônico/química , Sistema Hematopoético , Ácidos Hexurônicos/química , Humanos , Imunofenotipagem , Coelhos , Fatores de TempoRESUMO
The study of myelodysplastic syndromes (MDS) in murine models has now indicated the possible involvement of the bone marrow microenvironment in the generation of dysplastic hematopoietic cells. However, there is scant work on patient samples and the role of hypomethylating agents on the bone marrow stromal cells of MDS patients is unclear. We show that human MDS-MSCs exhibit phenotypic, transcriptomic and epigenetic abnormalities. Stimuli provided by MDS-MSCs impaired the growth and function of healthy HSPCs, which is further sustained autonomously in HSPCs for significant periods of time resulting in a failure for active hematopoietic engraftment across primary and secondary transplant recipients (chimerism: 0.34-91% vs 2.78%, engraftment frequencies: at 0.06 ± 0.02 vs full engraftment for MDS-MSC vs healthy groups, respectively). Hypomethylation of MDS-MSCs improved overall engraftment in most of the MDS-MSC groups tested (2/7 with p < 0.01, 3/7 with p < 0.05 and 2/7 with no significant difference). MDS-MSCs that fail to respond to hypomethylating therapy are associated with patients with rapid adverse disease transformation and this further suggests that MDS-MSCs may be an integral part of disease progression and have prognostic value as well as potential as a therapeutic target.
Assuntos
Azacitidina/farmacologia , Biomarcadores Tumorais/metabolismo , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Proliferação de Células , Epigênese Genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In the original version of this article there was a mistake in the spelling of the author Sujoy Ghosh, originally spelt Sujoy Gosh. This has now been corrected in both the PDF and HTML versions of the article.
RESUMO
To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 x 10(5) stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell-related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis-related antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Moreover, stem cell-related transcription factors, Nanog, Oct-4, Sox-2, and Rex-1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation potential after 25 passages.
Assuntos
Tecido Adiposo Branco/citologia , Células-Tronco Adultas/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Antígenos CD/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Calcificação Fisiológica , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Conexina 43/metabolismo , Meios de Cultura/farmacologia , Expressão Gênica/efeitos dos fármacos , Antígenos HLA-DR/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.
Assuntos
Antígenos CD34/metabolismo , Azóis/farmacologia , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Integrina alfa6/metabolismo , Células-Tronco/efeitos dos fármacos , Antígenos Thy-1/metabolismo , Animais , Células Cultivadas , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Imidazóis/farmacologia , Camundongos , Camundongos SCID , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/metabolismo , Relação Estrutura-AtividadeRESUMO
Rotating wall vessel (RWV) was used for the ex-vivo expansion of umbilical cord blood stem cells to meet the requirement of clinical application in the aspect of quantity and quality of the stem cells. The mononuclear cells (MNCs) from umbilical cord blood were cultured in T-flasks for 24 h, and then inoculated in RWV to culture for 200 h. The nucleated-cell numbers, pH and osmolality of the culture medium were determined every 24 h. The CD34+ cells content was measured and CFU-GM culture was carried out at 144 h and 197 h. Nucleated cells (NC) and CD34+ cells had a 435.5 +/- 87.6 fold expansion and a 32.7 +/- 15.6 fold expansion respectively in 197 h, and CFU-GM (colony-forming unit-granulocyte/macrophage) cells had a 21.7 +/- 4.9 fold expansion. In the whole course of culture, the pH and osmolality of the medium in the RWV were kept in the optimal hematopoietic stem cells' expansion conditions. pH was kept from 7.2 to 7.4, and the osmolality was kept from 290 mmol/kg to 310 mmol/kg. Owing to its structural particularity, the RWV could ensure cells to grow in the suspension state, could simulate the micro-environment of umbilical cord blood, and thus could make the hematopoietic stem cells expand largely in the RWV in short time.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/metabolismo , Proliferação de Células , Células Cultivadas , Meios de Cultura , Citocinas/farmacologia , Células-Tronco Hematopoéticas/fisiologia , HumanosRESUMO
Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24h. Flow cytometric assay for CD34+ cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 h. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5+/-87.6 times, and CD34+ cells 32.7+/-15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7+/-4.9 times. While in T-flasks, however, total cells density changed mildly, CD34+ cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Mecanotransdução Celular/fisiologia , Engenharia Tecidual/instrumentação , Contagem de Células , Técnicas de Cultura de Células/métodos , Proliferação de Células , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Estudos de Viabilidade , Humanos , Rotação , Engenharia Tecidual/métodosRESUMO
INTRODUCTION: Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system. METHODS: In this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer. RESULTS: Paradoxically, experiment results showed that the highest expansion of CD34+CD38-CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ-/-) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38-CD90+ and the in vivo competitive repopulating unit (CRU) functional assay. CONCLUSION: IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38-CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38-CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.