Unknown,Transcriptomics,Genomics,Proteomics

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Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

ABSTRACT: We used comparative transcriptome analysis to determine the relationship between rodent Multipotent Adult Progenitor Cells (MAPCs) and pluripotent embryonic stem cells (ESCs), or lineage restricted mesenchymal stem cells (MSCs) and neurospheres (NS). This comparison revealed a unique gene expression profile of MAPCs, that express transcripts of early endodermal and mesodermal but fewer ectodermal genes. In addition, MAPCs, but not MSCs or NS, express a number of genes expressed in ESCs including Oct-4 (Pou5f1) and other genes previously identified as ES cell associated transcripts and/or genes involved in maintenance of ESC selfrenewal capacity and pluripotency. However MAPCs do not express some key genes involved in maintaining selfrenewal and pluripotency in ESCs, including Nanog and Sox-2. Because MSC-like cells isolated under MAPC conditions are virtually identical to MSCs isolated in the presence of high serum, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. Finally, this unique molecular signature was seen irrespective of the microarray platform used, and was highly similar for both mouse and rat MAPCs. Keywords: cell type comparison design Two biological replicates were used for hybridizations. cRNA from MAPCs, ESCs, and NS were added to a universal reference cRNA.

ORGANISM(S): Mus musculus

SUBMITTER: Catherine Verfaillie

PROVIDER: E-GEOD-5947 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells.

Serafini Marta M Dylla Scott J SJ Oki Masayuki M Heremans Yves Y Tolar Jakub J Jiang Yuehua Y Buckley Shannon M SM Pelacho Beatriz B Burns Terry C TC Frommer Sarah S Rossi Derrick J DJ Bryder David D Panoskaltsis-Mortari Angela A O'Shaughnessy Matthew J MJ Nelson-Holte Molly M Fine Gabriel C GC Weissman Irving L IL Blazar Bruce R BR Verfaillie Catherine M CM

The Journal of experimental medicine 20070116 1

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and recon ...[more]

PMID: 17227908

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Project description:B-type lamins, the major structural component of the nuclear lamina, is thought to play a repressive role in the expression of many of its bound genes whose silencing are known to be critical for cell differentiation during early development. Using global mapping of lamin-B-chromatin interaction, we find that the binding to lamin-B indeed correlated with gene silencing in mouse embryonic stem cells (ESC) and ESC-derived trophectoderm (TE) lineage cells. To address whether lamin-B directly suppresses the expression of the bound genes, we have derived ESCs from lamin-B-null mouse blastocysts. Unexpectedly, microarray analyses of ESCs and TE cells show that B-type lamins do not repress the expression of their bound genes. Furthermore, the knockout mice that do not express any B-type lamins can develop to term but exhibit small body size and perinatal lethality. Our studies demonstrate that B-type lamins are not required for cell differentiation during early embryonic development. Instead, they are required for proper late embryonic and perinatal development. The availability of the null ESCs and mice opens the door to further define the functions of B-type lamins. The Dam and Dam-lamin B1 (Lmnb1) lentivirus generated from 293T cells were concentrated by spinning at 25,000 rpm with the SW28Ti rotor (Beckman) for 3 hours. The viral pellets were resuspended in 1x PBS. For ESCs, freshly trypsinized ESCs were plated into a 6-well plate with 2 ml complete GMEM medium containing either Dam or Dam-Lmnb1 lentivirus, spin-infected at 700g for 45 min, and incubated for a total of 48 hours. For TE cells, TSCs were differentiated toward TE cells for 6 days. On day 6, the TE cells were transduced by spin-infection as above with a fresh TS medium containing either Dam or Dam-Lmnb1 lentivirus. The TE cells were incubated for 48 hours after viral transduction. The adenine-methylated DNAs were amplified from the genomic DNA by adaptor-mediated PCR. The amplified DNAs were labeled by the Dual color DNA labeling kit (Roche). The labeled DNAs were hybridized with 2.1M mouse whole-genome tiling arrays (NimbleGen, 05327911001), which covers non-repetitive regions in approximately 250 bp intervals, according to the manufacturer's recommendation. This submission represents the gene expression component of the study.

Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Individual transcription factors (TFs) (Ascl1, Smad7, and Nr2f1) were induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic TFs was repressed by doxycycline (Dox); thus, TFs were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of a transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then in NeuroCult neural differentiation medium. Cells marked as PSANCAM+ were enriched by magnetic microbeads with anty-PSA-NCAM antibody (Miltenyi Biotec) on day 6 of culturing, whereas remaining cells are marked as PSANCAM-. Both PSANCAM+ and PSANCAM- cells were then cultured for another 8 days (total 14 days in differentiation). A time course experiment with Ascl1 induction did not include cell enrichment procedure. RNA was extracted with either Trizol (invitrogen) or mirVana kit (Thermo Fisher Scientific).

Dataset Information

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

Publications

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells.

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