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1.
Mol Biol (Mosk) ; 53(5): 725-740, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661474

RESUMO

Human pluripotent stem cells, which include embryonic stem cells and induced pluripotent cells (iPSCs), are capable of unlimited division and differentiation into all cells of the body. These cells are considered as a potential source of various types of cells for transplantations. The use of autologous iPSCs is not potentially associated with immune rejection and does not require immunosuppression required for allogeneic grafts. However, the high cost of this technology and the duration of obtaining iPSCs and differentiated cells may limit the use of autologous iPSCs in clinical practice. In addition, full equivalence and immunological compatibility of autologous iPSCs and their derivatives have been repeatedly questioned. One approach to solving the problem of the immunological compatibility of allogeneic derivatives of iPSCs can be the establishment of cell lines with reduced immunogenicity. Differentiated derivatives of such iPSCs may be suitable for transplantation to any patient. This review discusses the strategies for evading immune surveillance in normal and tumor processes that can be used to establish stem cell lines with reduced immunogenicity.


Assuntos
Linhagem Celular/citologia , Linhagem Celular/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Humanos
2.
Nat Commun ; 10(1): 4357, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554807

RESUMO

Cell therapy products (CTP) derived from pluripotent stem cells (iPSCs) may constitute a renewable, specifically differentiated source of cells to potentially cure patients with neurodegenerative disorders. However, the immunogenicity of CTP remains a major issue for therapeutic approaches based on transplantation of non-autologous stem cell-derived neural grafts. Despite its considerable side-effects, long-term immunosuppression, appears indispensable to mitigate neuro-inflammation and prevent rejection of allogeneic CTP. Matching iPSC donors' and patients' HLA haplotypes has been proposed as a way to access CTP with enhanced immunological compatibility, ultimately reducing the need for immunosuppression. In the present work, we challenge this paradigm by grafting autologous, MHC-matched and mis-matched neuronal grafts in a primate model of Huntington's disease. Unlike previous reports in unlesioned hosts, we show that in the absence of immunosuppression MHC matching alone is insufficient to grant long-term survival of neuronal grafts in the lesioned brain.


Assuntos
Rejeição de Enxerto/imunologia , Doença de Huntington/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Complexo Principal de Histocompatibilidade/imunologia , Neurônios/transplante , Animais , Diferenciação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Modelos Animais de Doenças , Teste de Histocompatibilidade , Humanos , Doença de Huntington/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Neurônios/citologia , Neurônios/imunologia , Primatas , Ratos Nus , Transplante Autólogo
3.
PLoS One ; 14(5): e0216815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071196

RESUMO

Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used αß T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these αß T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed γδ T cell into iPSCs (γδ T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an "NK cell-promoting" protocol to differentiate γδ T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic γδ T cells endowed with an array of NKRs and thus designated as "γδ natural killer T (γδ NKT) cells" were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, γδ NKT cells may provide a potent "off-the-shelf" cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers.


Assuntos
Antígenos de Neoplasias/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Antígenos de Neoplasias/genética , Células HCT116 , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células K562 , Células MCF-7 , Células T Matadoras Naturais/patologia , Neoplasias/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/genética , Células THP-1
4.
EBioMedicine ; 42: 443-457, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30926422

RESUMO

BACKGROUND: Comparing non-inbred autologous and allogeneic induced pluripotent stem cells (iPSCs) and their secreted subcellular products among non-human primates is critical for choosing optimal iPSC products for human clinical trials. METHODS: iPSCs were induced from skin fibroblastic cells of adult male rhesus macaques belonging to four unrelated consanguineous families. Teratoma generativity, host immune response, and skin wound healing promotion were evaluated subsequently. FINDINGS: All autologous, but no allogeneic, iPSCs formed teratomas, whereas all allogeneic, but no autologous, iPSCs caused lymphocyte infiltration. Macrophages were not detectable in any wound. iPSCs expressed significantly more MAMU A and E of the major histocompatibility complex (MHC) class I but not more other MHC genetic alleles than parental fibroblastic cells. All topically disseminated autologous and allogeneic iPSCs, and their exosomes accelerated skin wound healing, as demonstrated by wound closure, epithelial coverage, collagen deposition, and angiogenesis. Allogeneic iPSCs and their exosomes were less effective and viable than their autologous counterparts. Some iPSCs differentiated into new endothelial cells and all iPSCs lost their pluripotency in 14 days. Exosomes increased cell viability of injured epidermal, endothelial, and fibroblastic cells in vitro. Although exosomes contained some mRNAs of pluripotent factors, they did not impart pluripotency to host cells. INTERPRETATION: Although all of the autologous and allogeneic iPSCs and exosomes accelerated wound healing, allogeneic iPSC exosomes were the preferred choice for "off-the shelf" iPSC products, owing to their mass-production, with no concern of teratoma formation. FUND: National Natural Science Foundation of China and National Key R&D Program of China.


Assuntos
Exossomos/metabolismo , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cicatrização/imunologia , Animais , Biomarcadores , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Transformação Celular Neoplásica/genética , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Macaca mulatta , Camundongos
5.
Biochem Biophys Res Commun ; 511(3): 711-717, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30827508

RESUMO

Immunogenicity of immature pluripotent stem cells is a topic of intense debate. Immunogenic antigens, which are specific in pluripotent states, have not been described previously. In this study, we identified glypican-3 (GPC3), a known carcinoembryonic antigen, as a pluripotent state-specific immunogenic antigen. Additionally, we validated the applicability of human leukocyte antigen (HLA)-class I-restricted GPC3-reactive cytotoxic T lymphocytes (CTLs) in the removal of undifferentiated pluripotent stem cells (PSCs) from human induced pluripotent stem cell (hiPSC)-derivatives. HiPSCs uniquely express GPC3 in pluripotent states and were rejected by GPC3-reactive CTLs, which were sensitized with HLA-class I-restricted GPC3 peptides. Furthermore, GPC3-reactive CTLs selectively removed undifferentiated PSCs from hiPSC-derivatives in vitro and inhibited tumor formation in vivo. Our results demonstrate that GPC3 works as a pluripotent state-specific immunogenic antigen in hiPSCs and is applicable to regenerative medicine as a method of removing undifferentiated PSCs, which are the main cause of tumor formation.


Assuntos
Glipicanas/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Diferenciação Celular , Linhagem Celular , Glipicanas/análise , Antígeno HLA-A2/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Neoplasias/imunologia
6.
Nat Rev Genet ; 20(7): 377-388, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30737492

RESUMO

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human-rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.


Assuntos
Doenças Transmissíveis/terapia , Doenças Genéticas Inatas/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Neoplasias/terapia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Quimera/genética , Quimera/imunologia , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/patologia , Descoberta de Drogas/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Terapia Genética/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/transplante , Modelos Animais , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/imunologia , Transplante de Tecidos/métodos , Transplante Heterólogo
7.
J Immunol ; 202(8): 2240-2253, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30796179

RESUMO

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele (Q0bolton ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0bolton-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0bolton-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0bolton-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0bolton-AAT bound IL-8 and leukotriene B4, comparable to healthy control M-AAT, and significantly decreased leukotriene B4-induced neutrophil adhesion (p = 0.04). Through a mechanism involving increased mRNA stability (p = 0.007), ataluren treatment of HEK-293 significantly increased Q0bolton-AAT mRNA expression (p = 0.03) and Q0bolton-AAT truncated protein secretion (p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0bolton-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.


Assuntos
Alelos , Códon sem Sentido , Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina , Adulto , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/imunologia , Fígado/metabolismo , Masculino , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/imunologia , Deficiência de alfa 1-Antitripsina/sangue , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/imunologia
8.
Exp Clin Transplant ; 17(Suppl 1): 31-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30777520

RESUMO

To avoid the ethical issues of embryonic stem cells, genome engineering has focused on inducible pluripotent stem cells, which can develop into all 3 germ layers. The ability to detect methylation patterns in these cells allows research into pluripotency markers. The recently developed CRISPR system has allowed widespread application of genome engineering techniques. The CRISPR-Cas9 system, a potent system for genome editing, can be used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering can be initiated: homologous or nonhomologous DNA repair by the Cas9 nuclease. Delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells can be accomplished via viral and nonviral delivery methods. Nonviral delivery includes lipid-mediated transfection and electroporation. It has become the most common and efficient in vitro delivery method for human pluripotent stem cells. The CRISPR-Cas9 system can be combined with inducible pluripotent stem cells to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. Knockouts can also be utilized to investigate epigenetic roles and targets, such as investigation of DNA methylation. CRISPR could be combined with human pluripotent stem cells to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways. The CRISPR-Cas9 system can also be used to create null or nucleasedead Cas9, which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion, RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Doenças Genéticas Inatas/terapia , Genoma Humano , Células-Tronco Pluripotentes Induzidas/transplante , Transplante de Células-Tronco/métodos , Animais , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Fenótipo , Transplante de Células-Tronco/efeitos adversos , Resultado do Tratamento
9.
Methods Mol Biol ; 1899: 25-40, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649763

RESUMO

Human induced pluripotent stem cells (iPSCs) are a potential source of blood cells for transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. The development of widely varying reprogramming methods has enabled us nowadays to obtain iPSCs even from a small number of antigen-specific T cells from patients. As these T-cell-derived iPSCs (T-iPSCs) carry TCR gene rearrangements in their genomic DNA, they are likely useful for producing antigen-specific T cells and for studying T-cell development. T-cell immunotherapy is potentially an effective therapeutic strategy against many types of cancers and viral infections. If antigen-specific T cells tailored against diseases and for patients can be easily obtained, T-cell immunotherapy should become a popular choice of therapy. Here, we show the in vitro way to guide T-iPSCs sequentially to yield hematopoietic stem/progenitor cells (HSPCs), T-lineage cells, and mature CD8 single-positive T cells. These in vitro-generated CD8+ T cells display antigen-specific cytotoxity and perform general T-cell functions. This novel protocol thus provides means to generate antigen-specific T cells as well as chances to study normal human lymphopoiesis. It may help identify, and then clear away, barriers to T-cell immunotherapy such as immunological tolerance and cell exhaustion. T-iPSCs can confer their juvenile status upon their descendant T cells during pluripotency reprogramming and redifferentiation. This phenomenon should help to eliminate T-cell exhaustion.


Assuntos
Antígenos CD8/imunologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos T/citologia , Humanos , Imunoterapia , Células-Tronco Pluripotentes Induzidas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia
10.
FEBS Lett ; 593(4): 386-394, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30609020

RESUMO

Mesothelial cells, which cover the surface of visceral organs and serous cavities in mammals, play a crucial role in preventing adhesion. We previously reported that primary mesothelial progenitor cells (MPCs) can not only prevent postoperative adhesion but also promote liver regeneration after hepatectomy. Induced pluripotent stem cells (iPSCs) have the potential to be used for regenerative medicine. Here, we have established a differentiation protocol for mouse iPSC-derived MPCs (miMPCs) via the exposure to defined factors, as well as purification using MPC-specific cell surface antigens. Furthermore, the miMPCs had the ability to suppress postoperative adhesion and facilitate liver regeneration. This is the first report highlighting the generation of functional miMPCs, which may offer potential for de novo cell therapy.


Assuntos
Células Epiteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Regeneração Hepática/efeitos dos fármacos , Células-Tronco/citologia , Aderências Teciduais/terapia , Animais , Antígenos de Superfície/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/imunologia , Epitélio/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Masculino , Camundongos , Transplante de Células-Tronco , Células-Tronco/imunologia
11.
J Clin Immunol ; 39(1): 106-111, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30635825

RESUMO

PURPOSE: We report normal neutrophil count in a mother, who carries the same ELANE mutation as her daughter with severe congenital neutropenia. We hypothesized that the mother possessed wild- and mutant-type clones and the wild-type clones could generate neutrophils, whereas the mutant clones could not. METHODS: We confirmed mutant variant ratio by sequence signals and measured the frequency of the mutant allele by subcloning in various cell types. We established the ELANE-mutated and non-mutated induced pluripotent stem cells (iPSCs) from the mother's T cells and compared granulopoiesis between these iPSCs. RESULTS: In the sequence analysis of isolated peripheral blood (PB), nail and hair, the mutant variant was detected in approximately 40-60% of lymphocytes, monocytes, hematopoietic progenitor cells, and hair as well as in a small percentage of nail, but in none of the neutrophils. In the subcloning analysis of extracted DNA from CD3+ and CD34+ cells, the mutant allele was identified in 37.5% and 38.1%, respectively. We reprogrammed the mother's PB cells and established the ELANE-mutated and non-mutated iPSCs. Granulopoiesis from mutated iPSCs revealed little sensitivity to granulocyte colony-stimulating factor in comparison with non-mutated iPSCs. CONCLUSIONS: These observations strongly suggest that mutant-carrying neutrophils did not appear in the mother's PB because mutated clones could not differentiate into neutrophils. The mother's normal hematological phenotype could be explained by the perseverance of normal, non-mutated granulopoiesis.


Assuntos
Elastase de Leucócito/genética , Mutação/genética , Alelos , Linhagem Celular , Pré-Escolar , Feminino , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Contagem de Leucócitos/métodos , Elastase de Leucócito/imunologia , Monócitos/imunologia , Mosaicismo , Mães , Mutação/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia
12.
Exp Mol Med ; 51(1): 3, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617277

RESUMO

Pluripotent stem cell transplantation is a promising regenerative strategy for treating intractable diseases. However, securing human leukocyte antigen (HLA)-matched donor stem cells is extremely difficult. The traditional approach for generating such cells is to establish homozygous pluripotent stem cell lines. Unfortunately, because of HLA diversity, this strategy is too time-consuming to be of practical use. HLA engineering of donor stem cells has been proposed recently as a means to evade graft-versus-host rejection in stem cell allotransplantation. This approach would be advantageous in both time and cost to the traditional method, but its feasibility must be investigated. In this study, we used CRISPR/Cas9 to knockout HLA-B from inducible pluripotent stem cells (iPSCs) with heterogenous HLA-B and showed that the HLA-B knockout iPSCs resulted in less immunogenicity in HLA-B antisera than that in the control. Our results support the feasibility of HLA-engineered iPSCs in stem cell allotransplantation.


Assuntos
Sistemas CRISPR-Cas , Antígenos HLA/genética , Histocompatibilidade , Células-Tronco Pluripotentes Induzidas/imunologia , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Camundongos , Camundongos Nus
13.
Am J Pathol ; 189(2): 339-353, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448404

RESUMO

Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of ß-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition-dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD.


Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Miócitos Cardíacos , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Cardiomiopatias/imunologia , Cardiomiopatias/patologia , Fibrose , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia
14.
Cancer Sci ; 110(1): 16-22, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30485606

RESUMO

Recent outstanding clinical results produced by engineered T cells, including chimeric antigen receptors, have already facilitated further research that broadens their applicability. One such direction is to explore new T cell sources for allogeneic "off-the-shelf" adoptive immunotherapy. Human pluripotent stem cells could serve as an alternative cell source for this purpose due to their unique features of infinite propagation ability and pluripotency. Here, we describe the current state of engineered T cell transfer with the focus on cell manufacturing processes and the potentials and challenges of induced pluripotent stem cell-derived T cells as a starting material to construct off-the-shelf T-cell banks.


Assuntos
Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Linfócitos T/transplante , Diferenciação Celular/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Neoplasias/imunologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante Homólogo
15.
EBioMedicine ; 39: 315-331, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30579862

RESUMO

BACKGROUND: Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models. METHODS: Here, we used an innovative, state-of-the-art iPSC-derived human retinal pigment epithelium (RPE) model to study ZIKV retinal impairment. FINDINGS: We showed that the human RPE is highly susceptible to ZIKV infection and that a ZIKV African strain was more virulent and led to a more potent epithelium disruption and stronger anti-viral response than an Asian strain, suggesting lineage differences. Moreover, ZIKV infection led to impaired membrane dynamics involved in endocytosis, organelle biogenesis and potentially secretion, key mechanisms of RPE homeostasis and function. INTERPRETATION: Taken together, our results suggest that ZIKV has a highly efficient ocular tropism, which creates a strong inflammatory environment that could have acute or chronic adverse effects. FUND: This work was funded by Retina France, REACTing and La Région Languedoc-Roussillon.


Assuntos
Interferons/metabolismo , Epitélio Pigmentado da Retina/virologia , Infecção por Zika virus/imunologia , Zika virus/patogenicidade , Células Cultivadas , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/virologia , Interferons/genética , Modelos Biológicos , Fagocitose , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/imunologia , Tropismo Viral , Replicação Viral , Zika virus/classificação , Zika virus/fisiologia , Infecção por Zika virus/genética , Infecção por Zika virus/virologia
16.
J Vis Exp ; (141)2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30531715

RESUMO

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.


Assuntos
Adesão Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos/metabolismo , Microfluídica/métodos , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Leucócitos/imunologia
17.
Sci Rep ; 8(1): 16550, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410112

RESUMO

The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches' potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells.


Assuntos
Antígenos CD/metabolismo , Epitélio Anterior/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD/genética , Antígenos de Superfície/genética , Caderinas/genética , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Epitélio Anterior/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Fator de Transcrição PAX6/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/imunologia , Proteoglicanas/genética , Análise de Célula Única
18.
Differentiation ; 104: 42-49, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30453197

RESUMO

Macrophages are phagocytic immune cells resident in every tissue that are not only important for host defence, but are also involved in tissue homeostasis, injury, and disease. Despite increasingly sophisticated methods for in vitro macrophage isolation, expansion and activation over the past three decades, these have largely been restricted to modelling bone-marrow or blood-derived cells. The in vitro derivation of macrophages from human pluripotent stem cells provides new opportunities to study macrophage biology, including the factors that impact human myeloid development and those that induce macrophage activation. While sharing many of the functional characteristics of monocyte-derived macrophages, stem cell-derived macrophages may offer new opportunities to understand the role of development or tissue context in innate immune cell function. Immune responsiveness to pathogenic challenge is known to be impacted by a macrophage's history of prior exposure, as well as ontogeny and tissue context. Therefore, we explore the factors of in vitro derivation likely to influence macrophage phenotype and function.


Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Células-Tronco Pluripotentes/citologia , Humanos , Imunidade Inata/genética , Células-Tronco Pluripotentes Induzidas/imunologia , Macrófagos/imunologia , Monócitos/citologia , Monócitos/imunologia , Células-Tronco Pluripotentes/imunologia
19.
Cell Stem Cell ; 23(6): 850-858.e4, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30449714

RESUMO

Limited T cell availability and proliferative exhaustion present major barriers to successful T cell-based immunotherapies and may potentially be overcome through the use of "rejuvenated" induced pluripotent stem cells derived from antigen-specific T cells (T-iPSCs). However, strict antigen specificity is essential for safe and efficient T cell immunotherapy. Here, we report that CD8αß T cells from human T-iPSCs lose their antigen specificity through additional rearrangement of the T cell receptor (TCR) α chain gene during the CD4/CD8 double positive stage of in vitro differentiation. CRISPR knockout of a recombinase gene in the T-iPSCs prevented this additional TCR rearrangement. Moreover, when CD8αß T cells were differentiated from monocyte-derived iPSCs that were transduced with an antigen-specific TCR, they showed monoclonal expression of the transduced TCR. TCR-stabilized, regenerated CD8αß T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies.


Assuntos
Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Células-Tronco Pluripotentes Induzidas/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/imunologia , Células Tumorais Cultivadas
20.
Methods Mol Biol ; 1842: 3-27, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196398

RESUMO

The seeming setbacks noted for stem cells underscore the need for experimental studies for safe and efficacious application to patients. Both clinical and experimental researchers have gained valuable knowledge on the characteristics of stem cells, and their behavior in different microenvironment. This introductory chapter focuses on adult mesenchymal stem cells (MSCs) based on the predominance in the clinic. MSCs can be influenced by inflammatory mediators to exert immune suppressive properties, commonly referred to as "licensing." Interestingly, while there are questions if other stem cells can be delivered across allogeneic barrier, there is no question on the ability of MSCs to provide this benefit. This property has been a great advantage since MSCs could be available for immediate application as "off-the-shelf" stem cells for several disorders, tissue repair and gene/drug delivery. Despite the benefit of MSCs, it is imperative that research continues with the various types of stem cells. The method needed to isolate these cells is outlined in this book. In parallel, safety studies are needed; particularly links to oncogenic event. In summary, this introductory chapter discusses several potential areas that need to be addressed for safe and efficient delivery of stem cells, and argue for the incorporation of microenvironmental factors in the studies. The method described in this chapter could be extrapolated to the field of chimeric antigen receptor T-cells (CAR-T). This will require application to stem cell hierarchy of memory T-cells.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Ensaios Clínicos como Assunto , Citocinas/metabolismo , Humanos , Imunomodulação , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Fenótipo , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/métodos , Células-Tronco/imunologia
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