Bert Dontje

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.

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1.

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.

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.

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.

Differential effects of recombinant human colony stimulating factor (rh G‐CSF) on stem cells in marrow, spleen and peripheral blood in mice

Summary Previously it has been hypothesized that the granulopoietic and erythropoietic lineages may compete for differentiating stem cells. According to this hypothesis one would expect that a stimulation of granulopoiesis by G‐CSF administration would lead to a reduction of the stem cell pool and be followed by a decline of erythropoietic progenitor numbers. In addition one would expect an enhanced response of granulopoiesis if G‐CSF administration were combined with suppression of erythropoiesis by red cell transfusion.

BMI1 collaborates with BCR-ABL in leukemic transformation of human CD34 + cells

The major limitation for the development of curative cancer therapies has been an incomplete understanding of the molecular mechanisms driving cancer progression. Human models to study the development and progression of chronic myeloid leukemia (CML) have not been established. Here, we show that BMI1 collaborates with BCR-ABL in inducing a fatal leukemia in nonobese diabetic/severe combined immunodeficiency mice transplanted with transduced human CD34(+) cells within 4-5 months. The leukemias were transplantable into secondary recipients with a shortened latency of 8-12 weeks. Clonal analysis revealed that similar clones initiated leukemia in primary and secondary mice. In vivo, transformation was biased toward a lymphoid blast crisis, and in vitro, myeloid as well as lymphoid long-term, self-renewing cultures could be established. Retroviral introduction of BMI1 in primary chronic-phase CD34(+) cells from CML patients elevated their proliferative capacity and self-renewal properties. Thus, our data identify BMI1 as a potential therapeutic target in CML.

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.

Efficient mobilization of haematopoietic progenitors after a single injection of pegylated recombinant human granulocyte colony-stimulating factor in mouse strains with distinct marrow-cell pool sizes

We have compared the efficacy of a single injection of SD/01, a newly engineered, pegylated form of recombinant human granulocyte colony stimulating factor (rhG‐CSF), with a single injection of glycosylated rhG‐CSF (Filgrastim). SD/01 was administered to regular and recombinant inbred strains of mice (AKR, C57L/J, DBA/2, C57BL/6, AKXL) known to have widely distinct marrow‐cell pool sizes and proliferation kinetics. A single injection of G‐CSF was unable to mobilize granulocyte–macrophage colony‐forming units (CFU‐GM). In sharp contrast, a single dose of SD/01 resulted in massive mobilization of progenitors and stem cells. Although all mice strains showed qualitatively similar mobilization responses, large interstrain differences remained. C57L and C57BL/6 mice mobilized relatively poorly, whereas AKR and DBA/2 mice showed threefold to tenfold superior responses. In order to explain these different phenotypes, we studied the effects of SD/01 in nine AKXL recombinant inbred strains, derived from well‐responding AKR and poorly responding C57L parental strains. The best predictor for SD/01 responsiveness in these strains was marrow cellularity prior to mobilization. Comparison of the AKXL strain distribution pattern for marrow cellularity with loci previously mapped in these strains showed complete concordance with Aat, a serine protease inhibitor mapping to chromosome 12.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.

Autonomous behavior of hematopoietic stem cells

Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated. We developed a model in which two genetically and phenotypically distinct stem cell populations coexist in a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/6 (B6) day-14 fetal liver cells. We determined the mobilization potential, proliferation, and frequency of D2 and B6 stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colony-stimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that the number of late appearing CAFC subsets within the D2 cell population was approximately threefold higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was approximately 10-fold higher than B6 LTRA numbers. Our data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes, indicating that stem cell properties are predominantly regulated cell-autonomously.