Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Wolfgang Wagner ◽  
Alexandra Ansorge ◽  
Ute Wirkner ◽  
Volker Eckstein ◽  
Christian Schwager ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Expression Profiles ◽  
Hematopoietic Progenitor Cells ◽  
Molecular Evidence ◽  
Cdna Clones ◽  
Human Umbilical Cord ◽  
Primitive Function ◽  
Hematopoietic Progenitor ◽  
Self Renewal

AbstractThe molecular mechanisms that regulate asymmetric divisions of hematopoietic progenitor cells (HPCs) are not yet understood. The slow-dividing fraction (SDF) of HPCs is associated with primitive function and self-renewal, whereas the fast-dividing fraction (FDF) predominantly proceeds to differentiation. CD34+/CD38– cells of human umbilical cord blood were separated into the SDF and FDF. Genomewide gene expression analysis of these populations was determined using the newly developed Human Transcriptome Microarray containing 51 145 cDNA clones of the Unigene Set-RZPD3. In addition, gene expression profiles of CD34+/CD38– cells were compared with those of CD34+/CD38+ cells. Among the genes showing the highest expression levels in the SDF were the following: CD133, ERG, cyclin G2, MDR1, osteopontin, CLQR1, IFI16, JAK3, FZD6, and HOXA9, a pattern compatible with their primitive function and self-renewal capacity. Furthermore, morphologic differences between the SDF and FDF were determined. Cells in the SDF have more membrane protrusions and CD133 is located on these lamellipodia. The majority of cells in the SDF are rhodamine-123dull. These results provide molecular evidence that the SDF is associated with primitive function and serves as basis for a detailed understanding of asymmetric division of stem cells.

NFκB-Mediated Up-Regulation of Transcription Factors Related to More Primitive State of Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1246-1246
Author(s):  
Rodrigo A. Panepucci ◽  
Lucila H.B. Oliveira ◽  
Dalila L. Zanette ◽  
Greice A. Molfetta ◽  
Rita C.V. Carrara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Real Time ◽  
Quantitative Pcr ◽  
Expression Profiles ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Primitive State

Abstract We have previously shown that a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells (HSPC) as compared to bone marrow (BM) CD34+ is a higher expression of transcription targets and components of the nuclear factor kappa B (NF-κB) pathway. NFKB2 and RELB are sub-units of the transcription factor (TF) that specifically mediates the constitutive NF-κB signaling pathway and their increased levels could be related with the primitive state of the newborn’s HSPC. However, BM and UCB CD34+ HSPC differ in their sub-population compositions, and a higher proportion of more primitive cells among the CD34+ cells could account for those differences. CD133 is a surface marker expressed on a more primitive sub-population of CD34+ cells that are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells. We used flow cytometry, oligonucleotide microarray gene expression profiling and real time quantitative PCR to better characterize immunomagnetically sorted CD34+ and CD133+ HSPC derived from BM and UCB. We found that UCB CD34+ cells contain a larger proportion of CD133+ cells (around 70%), differing from BM CD34+ cells (around 30%). Cluster analysis of the expression profiles, encompassing 10.000 genes, showed that UCB CD133+ are more similar to UCB CD34+ than to BM CD133+ cells. Furthermore, a statistically significant higher expression of NFKB2 and RELB was demonstrated by quantitative PCR on UCB CD133+ HSPC, compared to BM. Overall this indicates that despite distinct compositions of the cells from UCB or BM, UCB HSPC display intrinsic molecular differences related to their ontological age. The comparison of the gene expression profiles of the CD133+ with the CD34+ populations revealed the higher expression of many well known factors related to more primitive HSPC and hemangioblasts. In fact, TFs such as RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 and HOXB4 were all present at higher levels in CD133+ HSPC. In an attempt to identify a key TF that could be responsible for the expression of these important factors, we carried a promoter analysis for the set of highly expressed TF found in the CD133 cells. A frequency of TF binding sites significantly higher than the expected was observed for the NF-κB TFs, including potential NF-κB targets such as RUNX1, GATA3 and USF1. Measurements of GATA3, NFKB2 and RELB expression by real-time PCR showed a higher expression of the three genes in CD133+ samples (both from BM and UCB), as well as a correlation of the expression levels of NFkB2 and RELB with one another and with GATA3 (Sperman’s correlation), indicating that GATA3 could be, in fact, regulated by NF-κB. To further test this hypothesis, we used interference RNA (RNAi) against NFKB2 in HSPC. Levels of NFKB2, GATA3 and RELB (a known target of NFKB2/RELB dimmers) were down-modulated, in comparison with cells transfected with control RNAi. Taken together, our data indicates that constitutive NF-κB signaling may act up-regulating transcription factors related to a more primitive state of HSPC.

Separation of Hematopoietic Progenitor Cells from Human Umbilical Cord Blood

1993 ◽  
Vol 2 (2) ◽  
pp. 243-245 ◽  
Author(s):  
ARNON NAGLER ◽  
MARGO PEACOCK ◽  
MARISA TANTOCO ◽  
DONALD LAMONS ◽  
THOMAS B. OKARMA ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Progenitor Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Progenitor

Differential Expression of Mll-AF9 Up-Regulated Genes Correlates with Enhanced Self-Renewal of Hematopoietic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 733-733 ◽  
Author(s):  
Ashish R. Kumar ◽  
Wendy A. Hudson ◽  
Weili Chen ◽  
Rodney A. Staggs ◽  
Anne-Francoise Lamblin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Cell Types ◽  
Third Generation ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Third

Abstract In order to understand the pathophysiology of leukemia, we need to study the effects of leukemic oncogenes on the rare hematopoietic stem and progenitor cells. We investigated the self-renewal capabilities of the various hematopoietic cell types derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll-fusion gene under the control of the endogenous promoter present in every hematopoietic stem/progenitor cell. In methylcellulose cultures, we compared myeloid colony formation of Mll-AF9 cells to wild type progenitor populations over three generations of plating. In the first generation of plating, the Mll-AF9 common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) formed more colonies than the hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs). However, at the third generation of plating, colony numbers formed by Mll-AF9 HSCs and CLPs were significantly greater than those formed by CMPs and GMPs. By the third generation only occasional colonies were found in the wild type groups. These results demonstrate that while Mll-AF9 led to an increase in self-renewal of all 4 cell types studied, these effects were more pronounced in HSCs and CLPs. To identify the downstream genes that mediate the growth deregulatory effects of Mll-AF9, we compared gene expression profiles of Mll-AF9 derived cells to their wild type counterparts. To assess gene expression levels, we extracted RNA from wild type and Mll-AF9 HSCs, CLPs, CMPs and GMPs. We then amplified and labeled the RNA for analysis by Affymetrix murine 430 2.0 genome arrays. In an unsupervised analysis, the various Mll-AF9 cells clustered with their corresponding wild type counterparts, indicating that the expression of most genes was not significantly altered by Mll-AF9. To identify the genes that are differentially expressed in the Mll-AF9 derived cells, we performed a two-way ANOVA (with the genotype and cell type as the two variables) allowing for a false discovery rate of 10%. In this analysis, we found that 76 genes were up-regulated in all Mll-AF9 progenitor cells compared to their wild-type counterparts. This list included known targets of Mll-fusion proteins Hoxa5, Hoxa7, Hoxa9 and Hoxa10. Also included were Evi1 and Mef2c, two genes that have been implicated in promoting enhanced self-renewal of murine hematopoietic cells. Importantly, in wild type mice, these 6 genes were expressed at higher levels in HSCs and CLPs compared to CMPs and GMPs (average 3–25 fold). While we observed an average 2–10 fold increase in expression of these genes in all Mll-AF9 cell types compared to their respective wild type controls, the expression level was 3–8 fold higher in Mll-AF9 HSCs and CLPs compared to CMPs and GMPs. Thus, the expression of genes known to be intrinsically related to self-renewal is further enhanced as a result of the Mll-AF9 fusion gene. In conclusion, while activation of the Mll-AF9 genetic program and the resulting enhanced self-renewal occurs in all 4 cell types studied, these effects are greatest in HSCs and CLPs. Thus, HSCs and CLPs are likely to be more efficient than CMPs and GMPs in producing cellular expansion and targets for cooperating mutations resulting in leukemia.

Differential gene expression profiling of adult murine hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 488-498 ◽  
Author(s):  
In-Kyung Park ◽  
Yaqin He ◽  
Fangming Lin ◽  
Ole D. Laerum ◽  
Qiang Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Differential Gene Expression ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Cdna Clones ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differential Gene

Abstract Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

Gene Expression Analysis of Hematopoietic Progenitor Cells Identifies Dlg7as a Potential Stem Cell Gene

Stem Cells ◽  
2007 ◽  
Vol 25 (6) ◽  
pp. 1498-1506 ◽  
Author(s):  
Kristbjorn Orri Gudmundsson ◽  
Leifur Thorsteinsson ◽  
Olafur E. Sigurjonsson ◽  
Jonathan R. Keller ◽  
Karl Olafsson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cell Gene ◽  
Stem Cell Gene

scholarly journals Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 152-155 ◽  
Author(s):  
FG Schuening ◽  
R Storb ◽  
RB Stead ◽  
S Goehle ◽  
R Nash ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Average Rate ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Reductase Gene ◽  
Successful Transfer

Abstract Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU- GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

scholarly journals Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

1994 ◽  
Vol 179 (6) ◽  
pp. 1867-1875 ◽  
Author(s):  
S Z Zhou ◽  
S Cooper ◽  
L Y Kang ◽  
L Ruggieri ◽  
S Heimfeld ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Recombinant Virus ◽  
High Efficiency ◽  
Hematopoietic Progenitor Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Progenitor ◽  
Adeno Associated Virus

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

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