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Gerald de Haan

Publications and source records attributed to Gerald de Haan.

At least 19 recordsLinked to original sources
PubMed2006

A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning.

Several studies have suggested that the cyclin-dependent kinase (CDK) inhibitor p21 plays a crucial role in regulating hematopoietic stem and progenitor pool size. To allow assessment of long-term stem cell functioning in vivo, we have backcrossed a p21 null allele to C57BL/6 (B6) mice, the most commonly used mouse strain in hematopoietic stem cell research. In various in vitro assays, the homozygous deletion of the p21 allele did not affect the number of hematopoietic cells in B6 mice. Furthermore, the competitive repopulation ability was not different between p21-deficient and wild-type stem cells from both young and aged (20-month-old) mice. These results show that p21 is not essential for regulation of stem cell number in steady state. When proliferative stress was applied on p21-deficient stem cells by serial transplantation of 1,500 Lin(-)Sca-1(+)c-kit(+) (LSK) cells, again no detrimental effect was observed on cobblestone area-forming cell (CAFC) frequency and competitive repopulating ability. However, when bone marrow cells from mice that received 2 Gy of irradiation were transplanted, p21 deficiency resulted in a more than fourfold reduction in competitive repopulation index. Finally, we did not find major differences in cell cycle status and global gene expression patterns between LSK cells from p21-deficient and wild-type mice. Our findings indicate that the background of mice used for studying the function of a gene by genetic modification may determine the outcome. Cumulatively, our data fail to support the notion that p21 is essential for stem cell function during steady-state hematopoiesis, but may be relatively more important under conditions of cellular stress.

Animals↗
PubMed2006

Fibroblast growth factors as regulators of stem cell self-renewal and aging.

Organ and tissue dysfunction which is readily observable during aging results from a loss of cellular homeostasis and reduced stem cell self-renewal. Over the past 10 years, studies have been aimed at delineating growth factors that will sustain and promote the self-renewal potential of stem cells and support the expansion of primitive stem cells in vitro and in vivo. Recently, strong evidence is emerging indicating that fibroblast growth factors (FGFs) play a crucial role in stem cell maintenance. FGFs belong to a family of polypeptide growth factors that are involved in multiple functions including cell proliferation, differentiation, survival and motility. In this review, we discuss the regulatory role of FGFs on hematopoietic stem cells (HSCs), neural stem cells (NSCs) and embryonic stem (ES) cells in maintaining stem cell self-renewal. These findings are useful and important to further our knowledge in stem cell biology and for therapeutic approaches.

Aging↗
PubMed2006

Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche?

Many adult tissue stem cells, such as the cells of the hematopoietic system, gastrointestinal epithelium, brain, epidermis, mammary gland and lung have now been identified, all of them fulfilling a crucial role in supplying organisms with mature cells during normal homeostasis as well as in times of tissue generation or repair. Two unique features characterize adult stem cells: the ability to generate new pluripotent stem cells (to self-renew) and the ability to give rise to differentiated progeny that has lost its self-renewal capacity. Our understanding of the mechanisms that determine whether, where and when a stem cell will self-renew or differentiate is still limited, but recent advances have indicated that the stem cell microenvironment, or niche, provides essential cues that direct these cell fate decisions. Moreover, loss of control over these cell fate decisions might lead to cellular transformation and cancer. This review addresses the current understandings of the molecular mechanisms that regulate hematopoietic stem cell self-renewal in the niche and how leukemic transformation might change the dependency of leukemic stem cells on their microenvironment for self-renewal and survival.

Animals↗
PubMed2006

Fibroblast growth factor-1 and -2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures.

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.

Animals↗
PubMed2006

Cellular memory and hematopoietic stem cell aging.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation in order to sustain lifelong blood production and simultaneously maintain the HSC pool. However, there is clear evidence that HSCs are subject to quantitative and qualitative exhaustion. In this review, we briefly discuss several known aspects of the stem cell aging process, including DNA damage, telomere shortening, and oxidative stress. Besides these known players, there is increasing evidence that higher order chromatin structure, largely defined by the histone code and affecting transcriptional activity, is important. A model is suggested which describes how epigenetic regulation of gene transcription by modulation of the chromatin structure in stem cells can account for regulation of the aging program.

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PubMed2006

Selective expression of cholesterol metabolism genes in normal CD34+CD38- cells with a heterogeneous expression pattern in AML cells.

OBJECTIVE: To identify adenosine triphosphate-binding-cassette (ABC) transporters that are selectively expressed in normal and/or leukemic CD34+CD38- stem cells. METHODS: Microarray experiments on murine stem cells identified 13 ABC transporters with a high expression level. Corresponding human transporters were analyzed in normal CD34+CD38- and CD34+CD38+ bone marrow cells by quantitative reverse transcriptase polymerase chain reaction. RESULTS: Five ABC transporters, including ABCG1, were differentially expressed with a higher expression in CD34+CD38- cells. Besides ABCG1, known to be involved in cholesterol metabolism, expression of another major cholesterol transporter (ABCA1), some cholesterol metabolism genes (3-hydroxy-3-methyl-glutaryl-CoA reductase, low-density lipoprotein receptor), and the transcription factor controlling ABCA1 and ABCG1 expression, liver-X-receptor-alpha (LXR-alpha), were assessed. All these genes were predominantly expressed in the more primitive subpopulation, indicating a high rate of cholesterol metabolism and transport. Conversely in acute myeloid leukemia (AML), a heterogeneous expression pattern was found consisting of a considerably higher expression of particularly LXR-alpha in CD34+ cells and a reverse expression pattern in a subset of AML CD34+CD38+ cells. CONCLUSION: These data suggest an active cholesterol metabolism and efflux in normal CD34+CD38- cells, although a subgroup of AMLs potentially demonstrate a hyperactive cholesterol metabolism.

ADP-ribosyl Cyclase 1↗
PubMed2006

Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells.

PURPOSE OF REVIEW: The clinical use of hematopoietic stem cells, which produce all mature blood cell lineages in the circulation, is continuously increasing. Identification of genes and gene networks specifying either stemness or commitment will not only be of major relevance for a fundamental understanding of developmental biology, but also for the emerging fields of tissue engineering and regenerative medicine. Our appreciation of the transcriptional machinery that distinguishes stem cells from their nonstem cell progeny is, however, rudimentary. State-of-the art genome-wide tools are now becoming available to elucidate intrinsic properties of stem cells. Here, we review recent progress that has been made in this field. RECENT FINDINGS: Approaches to study stem cell-specific genes and gene networks include genetical genomics, mRNA and microRNA expression profiling of carefully selected cells, proteomics, chromatin studies using 'CHIP-on-chip' tools, genome-wide binding site analyses for transcription factors and chromatin-remodeling proteins, and tools to study the three-dimensional organization of gene loci. It is promising to see that the combined application of these tools has resulted in the identification of multiple novel genes that regulate stem cell self-renewal. SUMMARY: Exploitation of the available technology and integrating the data by translation into a dynamic model of networks, operating in all four dimensions, will be essential to fully comprehend the elusive concept of 'stemness'. It is time to harvest.

Animals↗
PubMed2006

Identification of quantitative trait loci regulating haematopoietic parameters in B6AKRF2 mice.

The haematopoietic system is a complex organised tissue with a hierarchical structure. Identification of organisational pathways within the haematopoietic system is relevant for a better understanding of haematopoiesis in health and disease. We have analysed numerous haematopoietic parameters in two panels of a total of 157 genetically distinct B6AKRF2 mice, derived from an intercross between AKR and C57Bl/6 mice, strains known to differ in various stem cell traits. The major objective of our study was to assess the extent to which various haematopoietic parameters, such as stem cell numbers, progenitor cell cycling, progenitor cell mobilisation and neutrophil numbers in blood and bone marrow are coregulated. The genotypes of these mice were used to search for genetic loci that regulate these parameters. We found significant quantitative trait loci (QTL) associated with the number of stem cells (CAFC-35) in the bone marrow and the number of neutrophils in the blood. However, most haematopoietic parameters appeared to be controlled by non-heritable (epigenetic) factors, or by multiple QTLs. Our study reveals striking differences in structure of the haematopoietic hierarchy between individual mice. Surprisingly, stem and progenitor cell pool size and proliferation rate, as well as peripheral blood cell counts are all independently regulated.

Animals↗
PubMed2005

The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion.

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.

Animals↗
PubMed2005

A statistical multiprobe model for analyzing cis and trans genes in genetical genomics experiments with short-oligonucleotide arrays.

Short-oligonucleotide arrays typically contain multiple probes per gene. In genetical genomics applications a statistical model for the individual probe signals can help in separating "true" differential mRNA expression from "ghost" effects caused by polymorphisms, misdesigned probes, and batch effects. It can also help in detecting alternative splicing, start, or termination.

Animals↗
PubMed2005

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'.

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

Carrier Proteins↗
PubMed2005

Administration of low-dose cytarabine results in immediate S-phase arrest and subsequent activation of cell cycling in murine stem cells.

OBJECTIVE: Hematopoietic stem cells (HSC) are considered to display a quiescent state with low turnover rate. We investigated the cell-cycle kinetics of HSC after a single dose of cytarabine (Ara-C). MATERIALS AND METHODS: We analyzed by flow cytometry the cell-cycle status of lin(low)sca-1(+)c-kit(+) (LSK) stem cells isolated from the bone marrow of C57Bl/6 mice sacrificed at 0, 2, 4, 6, 8, 12, 20, 48, 72, and 96 hours after intraperitoneal injection of Ara-C (100 mg/kg) using 7-aminoactinomycin-D (7-AAD) for DNA staining. In vivo bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in HCS were also measured. RESULTS: Two hours after administration of Ara-C, LSK cells ceased to incorporate BrdU. At 4 hours, a decrease of S-phase cells from 10% at baseline to 4% was found (p < 0.05), followed by a rapid increase of BrdU and 7-AAD incorporation reaching a maximum of 28% S-phase cells at 20 hours (p < 0.001). Ki-67 expression suggested recruitment of 20% of cells from G0 into cell cycle. The total number of LSK cells increased 2.5-fold within this short time interval. After 72 hours, a recovery of cell cycling to baseline levels was observed. CONCLUSION: This data shows that a single injection of Ara-C first rapidly induced S-phase arrest in HSC for up to 4 hours. Subsequently, an unexpectedly rapid activation of HCS with recruitment of G0 cells into cell cycle was observed. The mechanism of cell-cycle activation of LSK cells remains unknown, but reduction of the number of differentiated end cell did not appear to be the primary trigger.

Animals↗
PubMed2005

A genetic and genomic approach to identify longevity genes in mice.

Natural variation in rate of aging or longevity in mice and humans shows a very complex inheritance pattern. Few targeted genetic screens have identified longevity genes in mammals, partly as a result from the fact that the genetics of longevity can only be studied reliably in cohorts of mice. In this paper we propose that a combined genetic and genomic analysis of large families of fully genotyped recombinant inbred mice may provide a crucial tool to the aging research community. As a proof of principle we describe preliminary studies in which variation in gene expression patterns in hematopoietic stem cells and brain were genetically linked to longevity.

Animals↗
PubMed2005

Impaired hematopoietic stem cell functioning after serial transplantation and during normal aging.

Adult somatic stem cells possess extensive self-renewal capacity, as their primary role is to replenish aged and functionally impaired tissues. We have previously shown that the stem cell pool in short-lived DBA/2 (D2) mice is reduced during aging, in contrast to long-lived C57BL/6 (B6) mice. This suggests the existence of a genetically determined mitotic clock operating in stem cells, which possibly limits organismal aging. In the study reported here, unfractionated bone marrow (BM) cells or highly purified Lin(-)Sca-1(+)c-kit(+) (LSK) cells were serially transplanted in lethally irradiated D2 and B6 mice. In both strains, serial transplantation resulted in a substantial loss of stem cell activity. However, as we estimate that in B6 mice, the maximum number of population doublings of primitive cells is approximately 30, in D2 mice this is only approximately 20, resulting in a 1,000-fold difference in expansion potential, irrespective of whether whole bone marrow or purified hematopoietic stem cells (HSCs) were transplanted. Interestingly, recipients reconstituted with serially transplanted BM cells were able to accept a freshly isolated graft without any further conditioning. Finally, we show that whereas transplantation of BM cells into healthy, nonconditioned, young B6 recipients does not lead to engraftment, young BM cells do engraft and provide multilineage reconstitution in nonirradiated aged mice. Our data clearly establish the relevance of an intrinsic, genetically controlled program associated with impaired stem cell functioning during aging.

Animals↗
PubMed2004

Competitive clonal hematopoiesis in mouse chimeras explained by a stochastic model of stem cell organization.

Many current experimental results show the necessity of new conceptual approaches to understand hematopoietic stem cell organization. Recently, we proposed a novel theoretical concept and a corresponding quantitative model based on microenvironment-dependent stem cell plasticity. The objective of our present work is to subject this model to an experimental test for the situation of chimeric hematopoiesis. Investigating clonal competition processes in DBA/2-C57BL/6 mouse chimeras, we observed biphasic chimerism development with initially increasing but long-term declining DBA/2 contribution. These experimental results were used to select the parameters of the mathematical model. To validate the model beyond this specific situation, we fixed the obtained parameter configuration to simulate further experimental settings comprising variations of transplanted DBA/2-C57BL/6 proportions, secondary transplantations, and perturbation of stabilized chimeras by cytokine and cytotoxic treatment. We show that the proposed model is able to consistently describe the situation of chimeric hematopoiesis. Our results strongly support the view that the relative growth advantage of strain-specific stem cells is not a fixed cellular property but is sensitively dependent on the actual state of the entire system. We conclude that hematopoietic stem cell organization should be understood as a flexible, self-organized rather than a fixed, preprogrammed process.

Animals↗
PubMed2004

The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

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