ABSTRACT
It has been well established that histone and DNA modifications are critical to maintaining the equilibrium between pluripotency and differentiation during early embryogenesis. Mutations in key regulators of DNA methylation have shown that the balance between gene regulation and function is critical during neural development in early years of life. However, there have been no identified cases linking epigenetic regulators to aberrant human development and fetal demise. Here, we demonstrate that a homozygous inactivating mutation in the histone deacetylase SIRT6 results in severe congenital anomalies and perinatal lethality in four affected fetuses. In vitro, the amino acid change at Asp63 to a histidine results in virtually complete loss of H3K9 deacetylase and demyristoylase functions. Functionally, SIRT6 D63H mouse embryonic stem cells (mESCs) fail to repress pluripotent gene expression, direct targets of SIRT6, and exhibit an even more severe phenotype than Sirt6-deficient ESCs when differentiated into embryoid bodies (EBs). When terminally differentiated toward cardiomyocyte lineage, D63H mutant mESCs maintain expression of pluripotent genes and fail to form functional cardiomyocyte foci. Last, human induced pluripotent stem cells (iPSCs) derived from D63H homozygous fetuses fail to differentiate into EBs, functional cardiomyocytes, and neural progenitor cells due to a failure to repress pluripotent genes. Altogether, our study described a germline mutation in SIRT6 as a cause for fetal demise, defining SIRT6 as a key factor in human development and identifying the first mutation in a chromatin factor behind a human syndrome of perinatal lethality.
Subject(s)
Mutation/genetics , Sirtuins/genetics , Animals , Cell Differentiation/genetics , Embryoid Bodies , Embryonic Stem Cells , Fetal Death , Gene Expression/genetics , Humans , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
Chronic neuroinflammation is observed in HIV+ individuals on suppressive combination antiretroviral therapy (cART) and is thought to cause HIV-associated neurocognitive disorders. We have recently reported that expression of HIV intron-containing RNA (icRNA) in productively infected monocyte-derived macrophages induces pro-inflammatory responses. Microglia, yolk sac-derived brain-resident tissue macrophages, are the primary HIV-1 infected cell type in the central nervous system (CNS). In this study, we tested the hypothesis that persistent expression of HIV icRNA in primary human microglia induces innate immune activation. We established multiple orthogonal primary human microglia-like cell cultures including peripheral blood monocyte-derived microglia (MDMG) and induced pluripotent stem cell (iPSC)-derived microglia. Unlike MDMG, human iPSC-derived microglia (hiMG), which phenotypically mimic primary CNS microglia, were robustly infected with replication competent HIV-1, and establishment of productive HIV-1 infection and de novo viral gene expression led to pro-inflammatory cytokine production. Blocking of HIV-1 icRNA expression, but not multiply spliced viral RNA, either via infection with virus expressing a Rev-mutant deficient for HIV icRNA nuclear export or infection in the presence of small molecule inhibitor of CRM1-mediated viral icRNA nuclear export pathway, attenuated induction of innate immune responses. These studies suggest that Rev-CRM1-dependent nuclear export and cytosolic sensing of HIV-1 icRNA induces pro-inflammatory responses in productively infected microglia. Novel strategies targeting HIV icRNA expression specifically are needed to suppress HIV-induced neuroinflammation.
ABSTRACT
Although most genes are expressed biallelically, a number of key genomic sites--including immune and olfactory receptor regions--are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.
Subject(s)
Alleles , Cell Lineage , Gene Rearrangement, B-Lymphocyte, Light Chain/genetics , Immunoglobulin kappa-Chains/genetics , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/metabolism , Animals , Chromatin Immunoprecipitation , Clone Cells/cytology , Clone Cells/immunology , Clone Cells/metabolism , DNA Replication Timing , Female , Hematopoiesis , Humans , Immunoglobulin kappa-Chains/immunology , Male , Mice , Mice, Inbred BALB C , Models, Animal , Models, Immunological , Precursor Cells, B-Lymphoid/immunology , Stochastic ProcessesABSTRACT
In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
Subject(s)
Central Nervous System/pathology , Herpesvirus 1, Human/immunology , Induced Pluripotent Stem Cells/cytology , Toll-Like Receptor 3/deficiency , Astrocytes/immunology , Astrocytes/virology , Biomarkers , Cell Differentiation , Cell Lineage , Cell Separation , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/immunology , Central Nervous System/virology , Child , Disease Susceptibility , Encephalitis, Herpes Simplex/immunology , Encephalitis, Herpes Simplex/metabolism , Encephalitis, Herpes Simplex/pathology , Encephalitis, Herpes Simplex/virology , Herpesvirus 1, Human/pathogenicity , Humans , Immunity, Innate , Induced Pluripotent Stem Cells/virology , Interferons/immunology , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Neural Stem Cells/immunology , Neural Stem Cells/virology , Neurons/immunology , Neurons/pathology , Neurons/virology , Oligodendroglia/immunology , Oligodendroglia/pathology , Oligodendroglia/virology , Toll-Like Receptor 3/geneticsABSTRACT
BACKGROUND: Sickle cell anemia causes severe complications and premature death. Five common ß-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the ß-globin gene cluster. This is labor intensive, and error prone. METHODS: We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles. RESULTS: We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs (rs3834466, rs28440105, rs10128556, and rs968857) marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs. CONCLUSION: Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources.
Subject(s)
Anemia, Sickle Cell/genetics , Haplotypes , Polymorphism, Single Nucleotide , beta-Globins/genetics , Adolescent , Adult , Anemia, Sickle Cell/pathology , Child , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Pluripotent Stem Cells/metabolism , Young AdultABSTRACT
The mouse Shb gene coding for the Src Homology 2-domain containing adapter protein B has recently been placed in context of BCRABL1-induced myeloid leukemia in mice and the current study was performed in order to relate SHB to human acute myeloid leukemia (AML). Publicly available AML databases were mined for SHB gene expression and patient survival. SHB gene expression was determined in the Uppsala cohort of AML patients by qPCR. Cell proliferation was determined after SHB gene knockdown in leukemic cell lines. Despite a low frequency of SHB gene mutations, many tumors overexpressed SHB mRNA compared with normal myeloid blood cells. AML patients with tumors expressing low SHB mRNA displayed longer survival times. A subgroup of AML exhibiting a favorable prognosis, acute promyelocytic leukemia (APL) with a PMLRARA translocation, expressed less SHB mRNA than AML tumors in general. When examining genes co-expressed with SHB in AML tumors, four other genes ( PAX5, HDAC7, BCORL1, TET1) related to leukemia were identified. A network consisting of these genes plus SHB was identified that relates to certain phenotypic characteristics, such as immune cell, vascular and apoptotic features. SHB knockdown in the APL PMLRARA cell line NB4 and the monocyte/macrophage cell line MM6 adversely affected proliferation, linking SHB gene expression to tumor cell expansion and consequently to patient survival. It is concluded that tumor SHB gene expression relates to AML survival and its subgroup APL. Moreover, this gene is included in a network of genes that plays a role for an AML phenotype exhibiting certain immune cell, vascular and apoptotic characteristics.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Leukemia, Myeloid, Acute/pathology , Proto-Oncogene Proteins/biosynthesis , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/genetics , Adult , Aged , Biomarkers, Tumor/analysis , Female , Gene Expression Profiling , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Proportional Hazards Models , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/genetics , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Transcriptome , Young AdultABSTRACT
Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) ß-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Anemia, Sickle Cell/genetics , Fetal Hemoglobin/genetics , Haplotypes , Adolescent , Adult , Arabs/genetics , Carrier Proteins/genetics , Child , Child, Preschool , Female , Gene Expression , Humans , Male , Microfilament Proteins , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Polymorphism, Single Nucleotide , Receptors, Cell Surface/genetics , Repressor Proteins , White People/genetics , Young Adult , beta-Globins/geneticsABSTRACT
Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.
Subject(s)
Induced Pluripotent Stem Cells , Octamer Transcription Factor-3 , Polycomb Repressive Complex 2 , Polycomb-Group Proteins , Animals , Cell Differentiation , Cell Proliferation , DNA Methylation , Enhancer of Zeste Homolog 2 Protein , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Silencing , Histones/genetics , Histones/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Mice , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolismABSTRACT
Induced pluripotent stem cells (iPSCs) hold great promise for modeling human hematopoietic diseases. However, intrinsic variability in the capacities of different iPSC lines for hematopoietic development complicates comparative studies and is currently unexplained. We created and analyzed 3 separate iPSC clones from fibroblasts of 3 different normal individuals using a standardized approach that included excision of integrated reprogramming genes by Cre-Lox mediated recombination. Gene expression profiling and hematopoietic differentiation assays showed that independent lines from the same individual were generally more similar to one another than those from different individuals. However, one iPSC line (WT2.1) exhibited a distinctly different gene expression, proliferation rate, and hematopoietic developmental potential relative to all other iPSC lines. This "outlier" clone also acquired extensive copy number variations (CNVs) during reprogramming, which may be responsible for its divergent properties. Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential.
Subject(s)
Genetic Heterogeneity , Hematopoiesis/physiology , Induced Pluripotent Stem Cells/metabolism , Cell Differentiation , Cell Line , Cluster Analysis , DNA Copy Number Variations , Epigenesis, Genetic , Gene Expression Profiling , Gene Expression Regulation , Humans , Induced Pluripotent Stem Cells/cytology , Thrombopoiesis/geneticsABSTRACT
The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.
Subject(s)
Cell Differentiation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Animals , Apoptosis/drug effects , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Carbazoles/pharmacology , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Lineage/genetics , Cell Proliferation/drug effects , Cytochrome P-450 CYP1B1 , Erythroid Cells/cytology , Erythroid Cells/drug effects , Erythroid Cells/metabolism , Feeder Cells/cytology , Feeder Cells/drug effects , Feeder Cells/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Genome, Human/genetics , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/enzymology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Megakaryocytes/cytology , Megakaryocytes/drug effects , Megakaryocytes/metabolism , Mice , Receptors, Aryl Hydrocarbon/agonistsABSTRACT
Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.
Subject(s)
Neovascularization, Physiologic/genetics , Transcription Factors/metabolism , Transcription, Genetic , Animals , Animals, Newborn , Cell Line , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/growth & development , Extracellular Matrix/metabolism , GATA2 Transcription Factor/metabolism , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/deficiency , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/physiology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Retinal Vessels/growth & development , Retinal Vessels/metabolism , Signal Transduction , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors, TFII/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
The derivation of patient-specific pluripotent cell lines through the introduction of a few transcription factors into somatic cells has opened new avenues for the study and treatment of human disorders. Induced pluripotent stem cells (iPSCs) and their derivatives offer a unique platform for disease modeling, drug discovery and toxicology, as well as an invaluable source of cells for regenerative therapies. Here, we provide an overview of the various strategies currently available for iPSC generation, highlighting recent advances and discussing some of the challenges faced in harnessing the true potential of iPSCs for biomedical research and therapeutic applications.
Subject(s)
Cell Engineering/methods , Induced Pluripotent Stem Cells/physiology , Transcription Factors/metabolism , Animals , Cell Differentiation/genetics , Cells, Cultured , Genetic Vectors , Humans , Induced Pluripotent Stem Cells/cytology , Mice , Models, Biological , Rats , Transcription Factors/geneticsABSTRACT
Human adipocytes express high levels of two distinct lipid droplet proteins, fat specific protein 27 (FSP27; also called CIDEC), a member of the CIDE family, and perilipin1 (PLIN1), a member of the PAT family. Both proteins play a role in fat metabolism in adipocytes, but how they interact is not known. Our present study demonstrates that FSP27 and PLIN1 co-localize and interact in cultured human primary adipocytes. We also found that the C-terminal domain of FSP27, aa 120-220, interacts with PLIN1. Individual expression of exogenous FSP27 or PLIN1 increased triglyceride content and decreased glycerol release (a measure of lipolysis), but co-expression of both proteins did not further increase triglyceride content or decrease lipolysis in human adipocytes. However, the combination of PLIN1 and FSP27 increased the average size of lipid droplets or caused the formation of unilocular adipocytes. Our data suggest that FSP27 interacts with PLIN1 to regulate lipid droplet size in human adipocytes in a concerted manner.
Subject(s)
Adipocytes/metabolism , Carrier Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , Triglycerides/metabolism , Apoptosis Regulatory Proteins , Cells, Cultured , Humans , Lipolysis , Perilipin-1 , Proteins/geneticsABSTRACT
Since the seminal discovery by Yamanaka et al. demonstrating that four transcription factors were capable of inducing nuclear reprogramming to a pluripotent state, a plethora of publications have followed aimed at improving the efficiency, simplicity, and safety of the original methodology that was based on the use of integrating retroviruses. A better understanding of the basic mechanisms behind reprogramming as well as an improvement in tissue culture conditions have allowed for the development of new tools based on different molecular approaches, such as excisable and nonintegrating vectors, RNA, proteins, and small compounds, among others. In most instances, a dynamic interplay exists between each method's efficiency of reprogramming versus overall safety, and these points need to be considered when choosing a particular approach. Regardless, the fast pace at which this field has advanced in recent years attracted many investigators to enter into the induced pluripotent stem cell (iPSC) world and has made the process of nuclear reprogramming and iPSC generation a routine lab technique.
Subject(s)
Cell Differentiation/genetics , Cellular Reprogramming/genetics , Genetic Vectors/metabolism , Induced Pluripotent Stem Cells/cytology , Transcription Factors/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Retroviridae/genetics , Transcription Factors/geneticsABSTRACT
Here, we describe a system for the exogenous control of gene expression in mammalian cells that relies on the control of translational termination. To achieve gene regulation, we modified protein-coding sequences by introduction of a translational termination codon just downstream from the initiator AUG codon. Translation of the resulting mRNA leads to potent reduction in expression of the desired gene product. Expression of the gene product can be controlled by treating cells that express the mRNA with either aminoglycoside antibiotics or several nonantibiotic compounds. We show that the extent of regulation of gene expression can be substantial (60-fold) and that regulation can be achieved in the case of a variety of different genes, in different cultured cell lines and in primary cells in vivo. This gene regulation strategy offers significant advantages over existing methods for controlling gene expression and should have both immediate experimental application and possible clinical application.
Subject(s)
Codon, Terminator/physiology , Gene Expression Regulation/physiology , Genetic Engineering/methods , Peptide Chain Termination, Translational/physiology , Acetanilides/pharmacology , Aminobenzoates/pharmacology , Aminoglycosides/pharmacology , Animals , Cell Line , Cells, Cultured , Genetic Vectors , Gentamicins/pharmacology , Luciferases/biosynthesis , Mice , Peptide Chain Termination, Translational/drug effects , Transgenes/geneticsABSTRACT
Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here, we demonstrate that the fetal lung is a potential source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells, accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal-lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43, and CD44. scRNA-seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFß/BMP, Notch, and YAP. Collectively, these data support the possibility that post-AGM development, functional HECs are present in the fetal lung, establishing this location as a potential extramedullary site of de novo hematopoiesis.
Subject(s)
Hemangioblasts , Hematopoiesis , Animals , Mice , Humans , Hematopoietic Stem Cells/metabolism , Cell Differentiation , Endothelium , Hemangioblasts/metabolismABSTRACT
A summit on cellular therapy for cancer discussed and presented advances related to the use of adoptive cellular therapy for melanoma and other cancers. The summit revealed that this field is advancing rapidly. Conventional cellular therapies, such as tumor infiltrating lymphocytes (TIL), are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Lymphocytes are being engineered to express high affinity T cell receptors (TCRs), chimeric antibody-T cell receptors (CARs) and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre-clinical models to be more effective than unselected populations and it is now possible to reprogram T cells and to produce T cells with stem cell characteristics. In the future, combinations of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of these therapies.
Subject(s)
Cell Transplantation/trends , Immunotherapy, Adoptive , Neoplasms/therapy , Cell Transplantation/methods , Cell Transplantation/statistics & numerical data , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/therapyABSTRACT
BACKGROUND: The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells (iPSCs) through the exogenous expression of transcription promises to revolutionize the study of human diseases. OBJECTIVE: Here we report on the generation of 25 iPSC lines from 6 patients with various forms of primary immunodeficiencies (PIDs) affecting adaptive immunity, innate immunity, or both. METHODS: Patients' dermal fibroblasts were reprogrammed by expression of 4 transcription factors, octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Krueppel-like factor 4 (KLF4), and cellular myelomonocytosis proto-oncogene (cMYC), by using a single excisable polycistronic lentiviral vector. RESULTS: iPSCs derived from patients with PIDs show a stemness profile that is comparable with that observed in human embryonic stem cells. After in vitro differentiation into embryoid bodies, pluripotency of the patient-derived iPSC lines was demonstrated by expression of genes characteristic of each of the 3 embryonic layers. We have confirmed the patient-specific origin of the iPSC lines and ascertained maintenance of karyotypic integrity. CONCLUSION: By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of iPSC lines from patients with PIDs represents a unique resource to investigate the pathophysiology of hematopoietic and extrahematopoietic manifestations of these diseases and might assist in the development of novel therapeutic approaches based on gene correction.
Subject(s)
Immunologic Deficiency Syndromes/pathology , Immunologic Deficiency Syndromes/physiopathology , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/physiology , Adaptive Immunity , Cell Dedifferentiation , Cell Differentiation , Cell Line , Cell Transdifferentiation , DNA/genetics , Gene Expression , Genes, myc , Humans , Immunity, Innate , Immunologic Deficiency Syndromes/genetics , Karyotyping , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Octamer Transcription Factor-3/genetics , Proto-Oncogene Mas , SOXB1 Transcription Factors/geneticsABSTRACT
A robust method of producing mature T cells from iPSCs is needed to realize their therapeutic potential. NOTCH1 is known to be required for the production of hematopoietic progenitor cells with T cell potential in vivo. Here we identify a critical window during mesodermal differentiation when Notch activation robustly improves access to definitive hematopoietic progenitors with T/NK cell lineage potential. Low-density progenitors on either OP9-hDLL4 feeder cells or hDLL4-coated plates favored T cell maturation into TCRab+CD3+CD8+ cells that express expected T cell markers, upregulate activation markers, and proliferate in response to T cell stimulus. Single-cell RNAseq shows Notch activation yields a 6-fold increase in multi-potent hematopoietic progenitors that follow a developmental trajectory toward T cells with clear similarity to post-natal human thymocytes. We conclude that early mesodermal Notch activation during hematopoietic differentiation is a missing stimulus with broad implications for producing hematopoietic progenitors with definitive characteristics.
Subject(s)
Induced Pluripotent Stem Cells , Mesoderm , Humans , Cell Lineage , T-Lymphocytes , Cell Differentiation , Receptors, NotchABSTRACT
Although mRNA vaccines against SARS-CoV-2 were highly efficacious against severe illness and hospitalization, they seem to be less effective in preventing infection months after vaccination, especially with the Delta variant. Breakthrough infections might be due to higher infectivity of the variants, relaxed protective measures by the general public in "COVID-19 fatigue", and/or waning immunity post-vaccination. Determining the neutralizing antibody levels in a longitudinal manner may address this issue, but technical complexity of classic assays precludes easy detection and quick answers. We developed a lateral flow immunoassay NeutraXpress™ (commercial name of the test kit by Antagen Diagnostics, Inc.) and tested fingertip blood samples of subjects receiving either Moderna or Pfizer vaccines at various time points. With this device, we confirmed the reported clinical findings that mRNA vaccine-induced neutralizing antibodies quickly wane after 3-6 months. Thus, using rapid tests to monitor neutralizing antibody status could help identify individuals at risk, prevent breakthrough infections, and guide social behavior to curtail the spread of COVID-19.