Hematopoietic stem cells engraft in mice with absolute efficiency

Benveniste, Patricia; Cantin, Claude; Hyam, Deborah; Iscove, Norman N.

doi:10.1038/ni940

Cited by 128 publications

(111 citation statements)

References 30 publications

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“…For each transcript, a single likely 3Ј transcript terminus was chosen by hand curation based on identification of clustered EST ends occurring closely downstream of a PAS (Table S2). The validity of each curated 3Ј terminus was tested by specific secondary PCR probing of total cDNA, initially amplified globally from murine ES cells or purified hematopoietic precursor cells (21) under stringent conditions of oligo(dT) primer annealing and reverse transcription (8). The global RT-PCR amplification procedure yields a mixture of individual cDNA fragments, each confined to a 300-to 500-nt window immediately upstream of a polyA sequence (8).…”

Section: Resultsmentioning

confidence: 99%

Identification of gene 3′ ends by automated EST cluster analysis

Muro

Herrington

Janmohamed

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The properties and biology of mRNA transcripts can be affected profoundly by the choice of alternative polyadenylation sites, making definition of the 3 ends of transcripts essential for understanding their regulation. Here we show that 22-52% of sequences in commonly used human and murine ''full-length'' transcript databases may not currently end at bona fide polyadenylation sites. To identify probable transcript termini over the entire murine and human genomes, we analyzed the EST databases for positional clustering of EST ends. The analysis yielded 58,282 murine-and 86,410 human-candidate polyadenylation sites, of which 75% mapped to 23,091 known murine transcripts and 22,891 known human transcripts. The murine dataset correctly predicted 97% of the 3 ends in a manually curated and experimentally supported benchmark transcript set. Of currently known genes, 15% had no associated prediction and 25% had only a single predicted termination site. The remaining genes had an average of 3-4 alternative polyadenylation sites predicted for each murine or human transcript, respectively. The results are made available in the form of tables and an interactive web site that can be mined for rapid assessment of the validity of 3 ends in existing collections, enumeration of potential alternative 3 polyadenylation sites of known transcripts, direct retrieval of terminal sequences for design of probes, and detection of polyadenylation sites not currently mapped to known genes.3Ј UTR ͉ gene prediction ͉ alternative polyadenylation ͉ transcriptome ͉ transcript probe design

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Section: Resultsmentioning

confidence: 99%

Identification of gene 3′ ends by automated EST cluster analysis

Muro

Herrington

Janmohamed

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, a method of calculations based on recurrence times for the Lorenz attractor (see Note) was developed by Gratrix and Elgin (2004) (Halsey and Jensen, 2004), suggesting the possibility to demonstrate the chaotic properties of a system, and perhaps even its precise quantitative nature. With the possibility of obtaining pure stem cells (Benveniste et al, 2003;Ikawa et al, 2004) and the capacity to monitor the dynamics of gene expression within single cells by timelapse video-microscopy (Janicki et al, 2004), it may not be so unrealistic to aim at predicting the unpredictable, HSC behavior for hematologists and the weather for Lorenz. Perhaps at a price, that of 'over'simplification.…”

Section: A Preview Of Things To Comementioning

confidence: 99%

“…In the same way as stars that are studied indirectly through their light, the latter reaching the observer with such delay that the source of light itself might have disappeared by the time it is captured, so were HSCs, invisible to our eyes and indirectly characterized by their progenies months or even years after commitment to differentiation has occurred, hence at a time when the cell of origin no longer exists. Thanks to ingenious approaches that started with quantitative and genetic analysis of spleen colonies (Becker et al, 1963), later developing into quantitative analysis of transplantation assays by limiting dilution or competitive repopulation (Harrison et al, 1988;Szilvassy et al, 1990), combined with improved gene transfer technology (Joyner et al, 1983;Williams et al, 1984) and the retroviral marking of HSCs (Dick et al, 1985;Jordan and Lemischka, 1990), as well as technological developments in flow cytometry, our capacity to understand and hence to directly visualize these cells has remarkably improved (Akashi et al, 2000; Benveniste et al, 2003;Matsuzaki et al, 2004). Identifying geographical niches (Calvi et al, 2003;Zhang et al, 2003;reviewed by Lemischka and Moore, 2003) in which to study HSCs in situ might provide insight into the regulation of asymmetrical division in these cells.…”

Section: Growth Factormentioning

confidence: 99%

The origin of hematopoietic cell type diversity

Hoang

2004

Oncogene

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“…The in vitro expansion of HSC from experimental animals has been enhanced (up to approximately forty times input) by the introduction of transgenes coding for MDR1 or HOXB4 (3,4), but HSC cannot currently be clonally expanded in vitro. Their ability to selfreplicate extensively in vivo is, however, undoubted because a single HSC is sufficient to repopulate the bone marrow of a recipient, which can then be used to repopulate the bone marrows of secondary recipients (5,6).…”

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Isolating gene-corrected stem cells without drug selection

Hatada

Arnold

Hatada

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drugresistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a ''purity'' of >30%, enriched >2,000-fold and with a recovery of Ϸ20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >3 days.electroporation ͉ flow cytometry ͉ fluorescent protein ͉ gene targeting ͉ gene therapy H ematopoietic stem cells (HSC) are among the longest known and best studied stem cells (1). They are characterized by their ability to fully repopulate the bone marrow of suitably prepared recipients and have been used therapeutically in the form of bone marrow transplants for several decades. HSC are, however, difficult to culture ex vivo without differentiating, and normal HSC have been induced to increase to only about six times the input number of HSC in vitro (2). The in vitro expansion of HSC from experimental animals has been enhanced (up to approximately forty times input) by the introduction of transgenes coding for MDR1 or HOXB4 (3, 4), but HSC cannot currently be clonally expanded in vitro. Their ability to selfreplicate extensively in vivo is, however, undoubted because a single HSC is sufficient to repopulate the bone marrow of a recipient, which can then be used to repopulate the bone marrows of secondary recipients (5, 6).The history of bone marrow transplantations clearly emphasizes that problems are likely to arise when there are histocompatibility differences between donors and recipients. Thus, the first successful bone marrow transplant was with an identical twin (genetically an autologous transplant) (7). Only the subsequent elucidation of histocompatibility antigens all...

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Hematopoietic stem cells engraft in mice with absolute efficiency

Cited by 128 publications

References 30 publications

Identification of gene 3′ ends by automated EST cluster analysis

Identification of gene 3′ ends by automated EST cluster analysis

The origin of hematopoietic cell type diversity

Isolating gene-corrected stem cells without drug selection

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