Global gene expression analysis of donor cells, low passage episomal stromal and non-stromal primed myeloid iPSC, and hESC
Ontology highlight
ABSTRACT: Global gene expression analysis of (a) human embryonic stem cells, (b) low passage episomal stromal-primed iPSC derived from growth-factor activated CD34+ myeloid progenitors from cord blood or GCSF mobilized peripheral blood, and (d) samples of donor fibroblasts or myeloid progenitors
Project description:Global gene expression analysis of FACS-purified CD31+CD146+ vascular progenitors (VP) derived from (a) human embryonic stem cells (VP-hESC), (b) adult fibroblast derived induced pluripotent stem cells (VP-AdF-iPSC), (c) stromal primed cord blood CD34+ myeloid progenitor derived iPSC (VP-sp-CB-iPSC), (d) corresponding starting fibroblasts and myeloid progenitors, (e) human umbilical vein endothelial cells, and (f) human dermal microvascular endothelial cells. Total RNA was harvested from (a) adult fibroblasts, (b) human myeloid progenitors: Non-nucloefected CD34+ CB cells that were expanded with growth factors from Day -3 until Day 0 and harvested, (c) FACS-purified CD31+CD146+ vascular progenitors (VP) that were differentiated from human embryonic stem cells (hESC), (d) VP differentiated from low passage stromal-primed episomal iPSC derived from growth-factor activated cord blood myeloid progenitors, (e) VP differentiated from iPSC derived from adult fibroblasts, (f) human umbilical vein endothelial cells, and (g) human dermal microvascular endothelial cells.. Illumina HumanHT-12 V4.0 expression beadchips were used for all analyses in this series. Three or independent samples of each sample type were each run on individual microarrays.
Project description:Analysis of the episomal backbone's influence on gene expression. The first hypothesis tested in the present study is that the episomal EBNA vectors, which rely on the EBNA-1 oncoprotein for episomal maintenance, have a greater influence on the cells' expression profiles than S/MAR vectors. The second hypothesis tested was that when bacterial sequences are removed from the episomal vector backbone, the gene disturbance is minimal.
Project description:T cells serve as pivotal effectors of the immune system and can be harnessed as therapeutic agents for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and mature T cells from CD34+ cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induced an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+ T cells. Single-cell RNA sequencing of human PSC-derived proT cells revealed a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrated efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.
Project description:Non-structural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication and is considered an attractive target for the development of novel antiviral drugs. We were initially interested in the interplay between nsp13 and interferon signaling, and found that nsp13 inhibited reporter signal of IFN-β promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-β promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted expression of foreign genes originated from plasmid transfection, but failed to inhibit them after chromosome integration. These data together with results from run-off transcription assay and RNA sequencing suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to episomal DNA transcriptional inhibition, And this restriction required direct interaction with episomal DNA. Further, we developed a high-throughput nsp13 drug screening method based on the correlation between the helicase activity and nsp13 inhibition on episomal DNA. This method evaluates the inhibitory effect of compounds on nsp13 by detecting the expression of reporter plasmids after co-transfection with nsp13 plasmids, which is economical and convenient compared with conventional methods. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs.
Project description:Collombet2016 - Lymphoid and myeloid cell
specification and transdifferentiation
This model is described in the article:
Logical modeling of lymphoid
and myeloid cell specification and transdifferentiation
Samuel Collombet, Chris van Oevelen,
Jose Luis Sardina Ortega, Wassim Abou-Jaoudé, Bruno Di
Stefano, Morgane Thomas-Chollier, Thomas Graf, and Denis
Thieffry
Proceedings of the National Academy of
Sciences of the United States of America
Abstract:
Blood cells are derived from a common set of hematopoietic
stem cells, which differentiate into more specific progenitors
of the myeloid and lymphoid lineages, ultimately leading to
differentiated cells. This developmental process is controlled
by a complex regulatory network involving cytokines and their
receptors, transcription factors, and chromatin remodelers.
Using public data and data from our own molecular genetic
experiments (quantitative PCR, Western blot, EMSA) or
genome-wide assays (RNA-sequencing, ChIP-sequencing), we have
assembled a comprehensive regulatory network encompassing the
main transcription factors and signaling components involved in
myeloid and lymphoid development. Focusing on B-cell and
macrophage development, we defined a qualitative dynamical
model recapitulating cytokine-induced differentiation of common
progenitors, the effect of various reported gene knockdowns,
and the reprogramming of pre-B cells into macrophages induced
by the ectopic expression of specific transcription factors.
The resulting network model can be used as a template for the
integration of new hematopoietic differentiation and
transdifferentiation data to foster our understanding of
lymphoid/myeloid cell-fate decisions.
This model is hosted on
BioModels Database
and identified by:
MODEL1610240000.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
Project description:Global gene expression analysis of (a) human embryonic stem cells, (b) adult fibroblasts with and without nucleofection of SOKM, (c) CD34+ cord blood cells at various time points during induction of pluripotency with SOKM, with or without co-culture with bone marrow stromal cells (BMSC), and (d) resulting stromal primed and non-stromal primed cord blood CD34+ myeloid iPSC
Project description:αβT cell- and B cell-depleted HLA-haploidentical haematopoietic stem cell transplantation is a life-saving therapeutic option to treat patients with high-risk leukemia. The G-CSF treatment stimulates mobilization from the bone marrow to blood of hematopoietic stem cells (HSC), and this manipulated graft also contains mature donor-derived NK and γδT cells, both exerting graft-versus-leukemia activity and control of infections at early stages after transplantation. The G-CSF-induced mobilization in the donor causes relevant increases of different myeloid cells, including polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). PMN-MDSC are present in high proportions in the graft and exert a sharp inhibition on the effector functions of co-infused mature NK cells. Conversely, low frequencies of PMN-MDSCs are detected in the blood of non-mobilized healthy donors. We used microarray technology to identify possible differences in the transcriptional programme of PMN-MDSCs isolated from blood of G-CSF mobilized donors as compared to those of non-mobilized healthy individuals.
Project description:Human induced pluripotent stem (iPS) cells have previously been derived from somatic cells using viral vectors that integrate transgenes into the genome. Genomic integration, however, can allow persistent leaky expression of the transgenes and can create insertional mutations, thus limiting the utility of these cells for both research and clinical applications. Here, we describe the derivation of human iPS cells free of vector and transgene sequences using non-integrating oriP/EBNA1-based episomal vectors. The resulting iPS cells are similar to human embryonic stem (ES) cells in both proliferative and developmental potential. These results demonstrate that reprogramming of human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one important obstacle to the clinical applications of these cells. This SuperSeries is composed of the following subset Series:; GSE15175: Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 1.c); GSE15176: Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 4.a) Experiment Overall Design: Total 21 samples were analyzed to confirm the similarity of human iPS cells derived with episomal vectors with human ES cells, and a dissimilarity with fibroblasts. Experiment Overall Design: Refer to individual Series