Unknown,Transcriptomics,Genomics,Proteomics

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Global gene expression analysis of peripheral blood cells, pluripotent cell lines, and iPS-derived T cells

ABSTRACT: Exhaustion of antigen-specific T cells represents a major challenge to adoptive immunotherapy against many types of cancer and viral infection. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8+ T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells (T-iPSCs) were then redifferentiated into CD8+ T cells that had high proliferative capacity and elongated telomeres. To confirm that T-iPSCs were compatible to other embryonic stem cells (ESCs) but different from T cells, and that redifferentiated T cells were compatible to T cells but different from natural killer (NK) cells, we analyzed and compared the gene expression profiles of the samples by cDNA microarray. These analyses revealed that our T-iPSCs and redifferentiated T cells were essentially the same as their counterpart pluripotent stem cells and T cells, respectively. We generated human T-iPSC lines from peripheral blood T cells of healthy donors. To confirm that established T-iPSCs were typical iPSCs, the gene expression profile of one T-iPSC clone derived from a healthy person (TkT3V1-7) was compared to those of peripheral blood T cells (CD4+ T cell and CD8+ T cell) and ESCs (KhES3). We also established T-iPSCs (H254SeVT-3) from a T-cell clone (H25-#4) which was derived from an HIV-1-infected patient. H254SeVT-3 was redifferentitated into functional T cells (reT-1 and reT-2.1) in vitro. The gene expression profiles of reT-1 and reT-2.1 were compared to those of H25-#4 and peripheral blood NK cells to clarify that the redifferentiated T cells carried the same characteristics as the original T-cell clone.

ORGANISM(S): Homo sapiens

SUBMITTER: Toshinobu Nishimura

PROVIDER: E-GEOD-43136 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Innate memory phenotype (IMP) CD4+ T cells are non-conventional Î±Î² T cells exhibiting features of innate immune cells, characterized as CD44high and CD62Llow in periphery. It is recently reported by our group that bone marrow chimeric mice lacking thymic MHCI expression develop predominantly IMP CD8+ T cells, while those lacking hematopoietic MHCI develop predominantly naÃ¯ve CD8+ T cells. Here we perform hirarchical clustering analysis and found that CD4+ T cells share similar property: chimeras lacking thymic MHCII gave rise to predominantly CD4+ T cells that resemble IMP CD4+ T cells observed in WT mice, and vice versa, chimeras lacking hematopoietic MHCII had a majority of naÃ¯ve-like CD4+ T cells resembling naÃ¯veCD4+ T cells seen in WT mice. We used microarrays to compare the global programme of gene expression to determine whether the hematopoietic MHCII selected CD4+ T cells are IMP, and whether the thymic MHCII selected CD4+ T cells are naÃ¯ve CD4+ T cells as observed in WT mice. Through hierarchical clustering and analysis of global gene differential expression, we determined that hematopoietic MHCII dependent IMP CD4+ T cells generated from WT bone marrow transplanted into irradiated MHCII-/- recipients, resemble IMP CD4+ T cells in WT mice, while naÃ¯ve CD4+ T cells generated from MHCII-/- bone marrow transplanted into irradiated WT recipients, resemble naÃ¯ve CD4+ T cells in WT mice. Cell Sorting was performed using a Cytopeia Influx Cell Sorter. Chimeric IMP (CD45.1+TCRÎ²+CD4+CD44highCD62Llow) CD4+ T cells were sorted from splenocytes of CD45.1+WTâCD45.2+MHCII-/- chimeras (WM IMP CD4), and chimeric naÃ¯ve (CD45.2+TCRÎ²+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of CD45.2+MHCII-/- â CD45.1+WT chimeras (MW naÃ¯ve CD4) respectively, 8 weeks post transplantation. WT IMP (TCRÎ²+CD4+CD44highCD62Llow) and naÃ¯ve (TCRÎ²+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of 8-week old WT mice.

Project description:Genomic rearrangements leading to intragenic gene fusion are mainly found in some types of haematopoietic malignancies and sarcomas. Recently they have been described also in carcinomas such as the papillary thyroid histotype (60%-70%) and the Hürthle thyroid tumours (58%). The presence of junction oncogene constitutes an area of exciting research for emerging therapy as targeting the RET-PTC1 fusion oncogene by using small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. Therefore, we developed a siRNA against RET-PTC1 junction and assess its efficiency on the human papillary thyroid carcinoma cell line TPC-1 which spontaneously harbours the RET-PTC1 oncogene. The targeted genes are assessed by microarray analysis by comparing the regulated genes by the siRNA_RET-PTC1 vs a siRNA_RET developed on the RET part of the mRNA minus the siRNA_control that contain four mutation within the RET-PTC1 sequence. To test the targeted genes in the TPC-1 cell line that spontaneously harbours RET-PTC1 junction of two siRNAs developed: Within the RET-PTC1 junction, and in the mRNA RET part. A non-specific siRNA harbouring 4 mutations within the RET-PTC1 sequence was used as negative control (siRNA_control). By real-time PCR (Q-RT-PCR) we demonstrated that both siRNAs (siRNA_RET-PTC1 and siRNA_RET) significantly reduce RET mRNA levels of about 85 % in TPC-1 cells. The negative control did not show an effect of RET mRNA levels. Three independent transfections were performed on TPC-1 cells using 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Cergy-Pontoise, France) and 50nM of i) siRNA_RET-PTC1 or ii) siRNA_RET or iii) siRNA_control that harbour 4 mutations within its sequence. Total RNAs of untreated cells and transfected cells were purified using the RNA cleanup and concentration kit (QIAGEN, Hilden, Germany) and gathered in 4 pools : 1) TPC-1 harbouring RET-PTC1 ; 2) TPC-1 silenced for RET-PTC1 with siRNA RET-PTC1 ; 3) TPC-1 silenced for siRNA RET ; 4) TPC-1 treated with the siRNA control.

Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates

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Global gene expression analysis of peripheral blood cells, pluripotent cell lines, and iPS-derived T cells

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