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1.
Nat Commun ; 12(1): 5761, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599177

RESUMO

The thymus is a central lymphoid organ primarily responsible for the development of T cells. A small proportion of B cells, however, also reside in the thymus to assist negative selection of self-reactive T cells. Here we show that the thymus of human neonates contains a consistent contingent of CD138+ plasma cells, producing all classes and subclasses of immunoglobulins with the exception of IgD. These antibody-secreting cells are part of a larger subset of B cells that share the expression of signature genes defining mouse B1 cells, yet lack the expression of complement receptors CD21 and CD35. Data from single-cell transcriptomic, clonal correspondence and in vitro differentiation assays support the notion of intrathymic CD138+ plasma cell differentiation, alongside other B cell subsets with distinctive molecular phenotypes. Lastly, neonatal thymic plasma cells also include clones reactive to commensal and pathogenic bacteria that commonly infect children born with antibody deficiency. Thus, our findings point to the thymus as a source of innate humoral immunity in human neonates.


Assuntos
Diferenciação Celular , Plasmócitos/citologia , Timo/citologia , Adulto , Antígenos CD/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Recém-Nascido , Subpopulações de Linfócitos/citologia , Análise de Componente Principal , RNA-Seq , Análise de Célula Única , Hipermutação Somática de Imunoglobulina/genética , Transcriptoma/genética
2.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575977

RESUMO

Amidst the global shortfalls in blood supply, storage limitations of donor blood and the availability of potential blood substitutes for transfusion applications, society has pivoted towards in vitro generation of red blood cells (RBCs) as a means to solve these issues. Many conventional research studies over the past few decades have found success in differentiating hematopoietic stem and progenitor cells (HSPCs) from cord blood, adult bone marrow and peripheral blood sources. More recently, techniques that involve immortalization of erythroblast sources have also gained traction in tackling this problem. However, the RBCs generated from human induced pluripotent stem cells (hiPSCs) still remain as the most favorable solution due to many of its added advantages. In this review, we focus on the breakthroughs for high-density cultures of hiPSC-derived RBCs, and highlight the major challenges and prospective solutions throughout the whole process of erythropoiesis for hiPSC-derived RBCs. Furthermore, we elaborate on the recent advances and techniques used to achieve cost-effective, high-density cultures of GMP-compliant RBCs, and on their relevant novel applications after downstream processing and purification.


Assuntos
Substitutos Sanguíneos/uso terapêutico , Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genética , Transfusão de Eritrócitos , Eritropoese/genética , Sangue Fetal/citologia , Humanos
3.
Nat Commun ; 12(1): 4706, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349112

RESUMO

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens.


Assuntos
Quimerismo , Feto/imunologia , Imunidade Materno-Adquirida/imunologia , Infecções/imunologia , Animais , Medula Óssea/metabolismo , Epigenoma , Feminino , Sangue Fetal/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Camundongos , Monócitos/citologia , Gravidez , Linfócitos T/citologia
4.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064452

RESUMO

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , Transgenes
5.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070163

RESUMO

Preeclampsia is associated with an increased cardiovascular morbidity of mother and offspring, thus contributing to a substantial burden in women and children's health. It has been proven that endothelial progenitor cell (EPC) numbers and functional characteristics are impaired in cardiovascular disease and preeclampsia, although causative factors for the latter have remained elusive. MicroRNA (miRNA) modifications are a potential mechanism through which exposure to an altered environment translates into the development of chronic disease. In this study, we examined whether development of preeclampsia corresponds to alterations of miRNAs in maternal- and cord-blood-derived EPC. To test this end, we analyzed maternal and neonatal miRNAs via RNA sequencing from endothelial cells of preeclamptic and healthy controls in different cell culture passages. We were able to demonstrate differentially represented miRNAs in all groups. Hsa-miR-1270 showed significantly different levels in cord blood EPC from preeclampsia versus control and was negatively correlated with mRNA levels of its predicted targets ANGPTL7 and TFRC. Transfection with an hsa-miR-1270 inhibitor decreased the tube formation capacity and chemotactic motility but did not change proliferation in vitro. Target predictions and gene set enrichment analyses identified alternative splicing as a significantly enriched pathway for hsa-miR-1270. The top miRNAs in three other groups were predicted to target transcriptional and developmental pathways. Here, we showed for the first time significantly different levels of miRNAs and differently represented mRNA levels of predicted target genes in EPC derived from preeclampsia. Understanding the effects of preeclampsia on the epigenetic mechanisms of EPC will be crucial and may provide initial insights for further evaluation of the benefits of therapies targeting this cell population.


Assuntos
Células Progenitoras Endoteliais/metabolismo , MicroRNAs/genética , Pré-Eclâmpsia/genética , Adulto , Proteínas Semelhantes a Angiopoietina/genética , Antígenos CD/genética , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Quimiotaxia , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Masculino , MicroRNAs/sangue , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , RNA Mensageiro/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Transferrina/genética , Adulto Jovem
6.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072923

RESUMO

Human umbilical cord blood (UCB) represents a valuable source of hematopoietic stem cells, particularly for patients lacking a matching donor. UCB provides practical advantages, including a lower risk of graft-versus-host-disease and permissive human leukocyte antigen mismatching. These advantageous properties have so far been applied for stem cell, mesenchymal stromal cell, and chimeric antigen receptor T cell therapies. However, UCB-derived professional antigen-presenting cells are increasingly being utilized in the context of immune tolerance and regenerative therapy. Here, we review the cell-specific characteristics as well as recent advancements in UCB-based cell therapies focusing on dendritic cells, monocytes, B lymphocytes, innate lymphoid cells, and macrophages.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Hematopoéticas/imunologia , Imunidade Inata/genética , Células Apresentadoras de Antígenos/transplante , Linfócitos B/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/citologia , Sangue Fetal/transplante , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos
7.
J Vis Exp ; (171)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34096918

RESUMO

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.


Assuntos
Antígenos CD34 , Plaquetas , Células-Tronco Hematopoéticas , Megacariócitos , Antígenos CD34/sangue , Plaquetas/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Megacariócitos/citologia
8.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063174

RESUMO

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-ß) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.


Assuntos
Sangue Fetal/citologia , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Células Th1/citologia , Células Th1/metabolismo , Apoptose , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Citocinas , Humanos , Células Th2/citologia , Células Th2/metabolismo
9.
Methods Mol Biol ; 2324: 339-360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165725

RESUMO

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Pseudogenes , Sequência de Bases , Biomarcadores Tumorais/genética , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/genética , Meios de Cultura , Meios de Cultivo Condicionados , DNA/sangue , DNA/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , DNA de Cadeia Simples/sangue , Células Progenitoras Endoteliais/citologia , Sangue Fetal/citologia , Glioblastoma/patologia , Humanos , Mutagênese Insercional , Proteína Homeobox Nanog/genética , Neoplasias/genética , Células-Tronco Neurais/citologia , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Homologia de Sequência do Ácido Nucleico , Transfecção , Células Tumorais Cultivadas
10.
Gac Med Mex ; 157(1): 29-34, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34125810

RESUMO

INTRODUCTION: Analysis of several parameters is required for adequate quality control in umbilical cord blood units (UCBU) when used for therapeutic purposes. OBJECTIVE: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. METHODS: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the sequence-specific priming technique. RESULTS: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY/11 and GP5/GP6+. CONCLUSIONS: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.


Assuntos
DNA Viral/isolamento & purificação , Sangue Fetal/virologia , Genoma Viral , Células-Tronco Hematopoéticas/virologia , Papillomaviridae/isolamento & purificação , Adulto , Linhagem Celular , Criopreservação , Eletroforese em Gel de Ágar , Feminino , Sangue Fetal/citologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora) , Células HeLa , Teste de Histocompatibilidade , Humanos , Papillomaviridae/genética , Reação em Cadeia da Polimerase/métodos , Adulto Jovem
11.
Sci Rep ; 11(1): 11658, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079033

RESUMO

The therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.


Assuntos
Encefalomielite Autoimune Experimental/terapia , Vesículas Extracelulares/transplante , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-17/imunologia , Células-Tronco Mesenquimais/química , Animais , Peso Corporal , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Vesículas Extracelulares/imunologia , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/imunologia , Humanos , Interferon gama/genética , Interleucina-10/genética , Interleucina-17/genética , Interleucina-4/genética , Interleucina-4/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
Nat Immunol ; 22(6): 723-734, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958784

RESUMO

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.


Assuntos
Autorrenovação Celular/imunologia , Células-Tronco Hematopoéticas/fisiologia , Reconstituição Imune , Células-Tronco Multipotentes/fisiologia , Estresse Fisiológico/imunologia , Adulto , Animais , Autorrenovação Celular/genética , Células Cultivadas , Epigênese Genética/imunologia , Feminino , Sangue Fetal/citologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Hematopoese , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Separação Imunomagnética , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Nectinas/metabolismo , Cultura Primária de Células , RNA-Seq , Análise de Célula Única , Sirtuína 1/metabolismo , Estresse Fisiológico/genética , Transplante Heterólogo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
13.
Front Immunol ; 12: 631353, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34017325

RESUMO

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/fisiologia , Sangue Fetal/citologia , Doença Enxerto-Hospedeiro/prevenção & controle , Células-Tronco Mesenquimais/metabolismo , Animais , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/imunologia , Citocinas/farmacologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos NOD , Controle de Qualidade
14.
Front Immunol ; 12: 651399, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968049

RESUMO

Background: Cord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition. Patients and Methods: Here we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status. Results: MMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or -DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc. Conclusions: We have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.


Assuntos
Quimerismo , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células Matadoras Naturais/imunologia , Troca Materno-Fetal/imunologia , Adulto , Antígeno CD56/análise , Antígeno CD56/metabolismo , Separação Celular , Feminino , Sangue Fetal/imunologia , Citometria de Fluxo , Humanos , Recém-Nascido , Células Matadoras Naturais/metabolismo , Masculino , Idade Materna , Gravidez , Adulto Jovem
15.
Sci Rep ; 11(1): 10603, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011992

RESUMO

Human delta-like 1 (hDlk1) is known to be able to regulate cell fate decisions during hematopoiesis. Mesenchymal stromal cells (MSCs) are known to exhibit potent immunomodulatory roles in a variety of diseases. Herein, we investigated in vivo functions of hDlk1-hMSCs and hDlk1+hMSCs in T cell development and T cell response to viral infection in humanized NOD/SCID/IL-2Rγnull (NSG) mice. Co-injection of hDlk1-hMSC with hCD34+ cord blood (CB) cells into the liver of NSG mice markedly suppressed the development of human T cells. In contrast, co-injection of hDlk1+hMSC with hCD34+ CB cells into the liver of NSG dramatically promoted the development of human T cells. Human T cells developed in humanized NSG mice represent markedly diverse, functionally active, TCR V[Formula: see text] usages, and the restriction to human MHC molecules. Upon challenge with Epstein-Barr virus (EBV), EBV-specific hCD8+ T cells in humanized NSG mice were effectively mounted with phenotypically activated T cells presented as hCD45+hCD3+hCD8+hCD45RO+hHLA-DR+ T cells, suggesting that antigen-specific T cell response was induced in the humanized NSG mice. Taken together, our data suggest that the hDlk1-expressing MSCs can effectively promote the development of human T cells and immune response to exogenous antigen in humanized NSG mice. Thus, the humanized NSG model might have potential advantages for the development of therapeutics targeting infectious diseases in the future.


Assuntos
Antígenos/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Imunidade , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Antígenos CD34/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Sangue Fetal/citologia , Feto/embriologia , Herpesvirus Humano 4/imunologia , Humanos , Fígado/citologia , Fígado/embriologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Camundongos SCID
16.
Aging (Albany NY) ; 13(8): 11705-11726, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875618

RESUMO

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) can differentiate into all blood lineages to maintain hematopoiesis, wound healing, and immune functions. Recently, cobalt-chromium alloy casting implants have been used extensively in total hip replacements; however, cobalt nanoparticles (CoNPs) released from the alloy were toxic to HSCs and HPCs. We aimed to investigate the mechanism underlying the toxic effect of CoNPs on HSCs/HPCs and to determine the protective effect of selenomethionine (SeMet) against CoNPs in vitro and in vivo. Human and rat CD34+ HSCs/HPCs were isolated from cord blood and bone marrow, respectively. CoNPs decreased the viability of CD34+ HSCs/HPCs and increased apoptosis. SeMet attenuated the toxicity of CoNPs by enhancing the antioxidant ability of cells. The protective effect of SeMet was not completely abolished after adding H2O2 to abrogate the improvement of the antioxidant capacity by SeMet. SeMet and CoNPs stimulated ATM/ATR DNA damage response signals and inhibited cell proliferation. Unlike CoNPs, SeMet did not damage the DNA, and cell proliferation recovered after removing SeMet. SeMet inhibited the CoNP-induced upregulation of hypoxia inducible factor (HIF)-1α, thereby disrupting the inhibitory effect of HIF-1α on breast cancer type 1 susceptibility protein (BRCA1). Moreover, SeMet promoted BRCA1-mediated ubiquitination of cyclin B by upregulating UBE2K. Thus, SeMet enhanced cell cycle arrest and DNA repair post-CoNP exposure. Overall, SeMet protected CD34+ HSCs/HPCs against CoNPs by stimulating antioxidant activity and DNA repair.


Assuntos
Cobalto/toxicidade , Intoxicação por Metais Pesados/prevenção & controle , Células-Tronco Hematopoéticas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Selenometionina/farmacologia , Administração Oral , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Cobalto/administração & dosagem , Meios de Cultura/toxicidade , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Sangue Fetal/citologia , Intoxicação por Metais Pesados/etiologia , Intoxicação por Metais Pesados/patologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Substâncias Protetoras/uso terapêutico , Ratos , Selenometionina/uso terapêutico
17.
J Mater Chem B ; 9(13): 3047-3054, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33885666

RESUMO

Prenatal diagnostics holds great significance for pregnant women desiring healthy babies. Fetal nucleated red blood cells (fNRBCs), bearing the complete genome of the fetus, have been regarded as an important biomarker for noninvasive prenatal diagnostics (NIPD). The high-performance detection and enrichment of fNRBCs from maternal blood, especially during early pregnancy, is urgently needed for NIPD, which, unfortunately, remains a big challenge for early-pregnancy fNRBC isolation. In this study, we developed an innovative platform based on silica microbeads for fNRBC isolation and release in early pregnancy. Microbeads were coated with self-assembled MnO2 nanoparticles (SiO2@MnO2) and then modified with a specific antibody. Benefiting from the three-dimensional nanostructure of the MnO2 nanoparticles, the isolation efficiency of the fNRBCs was enhanced. Subsequently, fNRBCs were released via dissolving the MnO2-nanoparticle coating using oxalic acid. We successfully isolated fNRBCs from the maternal peripheral blood samples of 20 pregnant women in the early pregnancy period, ranging from 41 to 62 gestational days. More importantly, the fetal origin of isolated cells was confirmed via fluorescent in situ hybridization and short tandem repeat analysis. This platform based on SiO2@MnO2 microbeads has verified the existence of fNRBCs in early-pregnancy maternal blood and is a promising approach for NIPD in early pregnancy.


Assuntos
Eritrócitos/citologia , Sangue Fetal/citologia , Microesferas , Nanoestruturas/química , Diagnóstico Pré-Natal , Feminino , Humanos , Compostos de Manganês/química , Óxidos/química , Gravidez , Dióxido de Silício/química
18.
Front Immunol ; 12: 652965, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912177

RESUMO

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.


Assuntos
Imunidade Adaptativa/fisiologia , Linfócitos T CD4-Positivos/imunologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Interferon beta/metabolismo , Transdução de Sinais/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Adulto , Fatores Etários , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Humanos , Separação Imunomagnética , Recém-Nascido , Cultura Primária de Células , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Front Immunol ; 12: 617592, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912153

RESUMO

Pregravid obesity has been shown to disrupt the development of the offspring's immune system and increase susceptibility to infection. While the mechanisms underlying the impact of maternal obesity on fetal myeloid cells are emerging, the consequences for T cells remain poorly defined. In this study, we collected umbilical cord blood samples from infants born to lean mothers and mothers with obesity and profiled CD4 T cells using flow cytometry and single cell RNA sequencing at resting and following ex vivo polyclonal stimulation. We report that maternal obesity is associated with higher frequencies of memory CD4 T cells suggestive of in vivo activation. Moreover, single cell RNA sequencing revealed expansion of an activated subset of memory T cells with maternal obesity. However, ex vivo stimulation of purified CD4 T cells resulted in poor cytokine responses, suggesting functional defects. These phenotypic and functional aberrations correlated with methylation and chromatin accessibility changes in loci associated with lymphocyte activation and T cell receptor signaling, suggesting a possible link between maternal obesogenic environment and fetal immune reprogramming. These observations offer a potential explanation for the increased susceptibility to microbial infection in babies born to mothers with obesity.


Assuntos
Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epigênese Genética , Sangue Fetal/citologia , Imunofenotipagem , Montagem e Desmontagem da Cromatina , Citocinas/metabolismo , Metilação de DNA , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Obesidade Materna/sangue , Obesidade Materna/genética , Obesidade Materna/imunologia , Obesidade Materna/metabolismo , Gravidez , Análise de Célula Única/métodos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma
20.
Front Immunol ; 12: 657261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33927724

RESUMO

The Warburg effect, defined as increased glycolysis and decreased oxidative phosphorylation, occurs in murine macrophages following LPS stimulation and is required for activation. There are differences between human and murine macrophage metabolic responses to stimulation, with peak metabolite concentrations occurring earlier in humans than mice. Complex changes occur in the human immune system with age, resulting in the very young and the very old being more susceptible to infections. Anti-bacterial immune responses in umbilical cord immune cells are considered deficient but there is a paucity of data on the role that metabolism plays. We hypothesized that metabolic responses in human macrophages occur early during activation. In addition, we hypothesized that umbilical cord derived macrophages have an altered immunometabolic response compared with adult macrophages. We demonstrate that adult and cord blood monocyte derived macrophages (MDM) immediately increase glycolysis in response to stimulation with LPS or Mycobacterium tuberculosis (Mtb), however only adult MDM decrease oxidative phosphorylation. At 24 hours post stimulation, glycolysis remains elevated in both adult and cord blood MDM, oxidative phosphorylation remains unchanged in the cord blood MDM and has normalized in the adult MDM stimulated with Mtb. However, LPS stimulated adult MDM have increased oxidative phosphorylation at 24 hours, illustrating differences in metabolic responses to different stimuli, time-dependent variation in responses and differences in macrophage metabolism in adults compared with umbilical cord blood. We compared the phenotype and function of macrophages derived from adult or cord blood. Cord blood MDM secreted less TNF following Mtb stimulation and more IL-6 following LPS stimulation compared with adult MDM. Our findings demonstrate that whilst cord blood MDM exhibit an immediate increase in glycolytic flux in response to stimulation, similar to adult MDM, cord blood MDM do not concomitantly decrease oxygen consumption. This indicates that adult macrophages shift to Warburg metabolism immediately after stimulation, but cord blood macrophages do not. Understanding the differences in the metabolic profiles of macrophages over a human lifetime will enable the translation of immunometabolism into effective immuno-supportive therapies that could potentially be targeted at vulnerable populations, such as the very old and the very young.


Assuntos
Sangue Fetal/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fatores Etários , Biomarcadores , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Glicólise , Humanos , Imunofenotipagem , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Fosforilação Oxidativa
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