Ronald van Os

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.

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo–expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self‐renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self‐renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.

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.

Regeneration of irradiated salivary glands with stem cell marker expressing cells

BACKGROUND Stem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patients quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland. METHODS Mouse submandibular gland tissue, isolated cells and cultured 3 day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15 Gy and transplanted with cells expressing defined markers. RESULTS Cells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function. CONCLUSIONS Different stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.

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.

Concise Review: Adult Salivary Gland Stem Cells and a Potential Therapy for Xerostomia

The ability to speak, swallow, masticate, taste food, and maintain a healthy oral cavity is heavily reliant on the presence of saliva, the hugely important effect of which on our everyday lives is often unappreciated. Hyposalivation, frequently experienced by people receiving radiation therapy for head and neck cancers, results in a plethora of symptoms whose combined effect can drastically reduce quality of life. Although artificial lubricants and drugs stimulating residual function are available to ameliorate the consequences of hyposalivation, their effects are at best transient. Such management techniques do not address the source of the problem: a lack of functional saliva‐producing acinar cells, resulting from radiation‐induced stem cell sterilization. Post‐radiotherapy stimulation of cell proliferation only results in improved saliva secretion when part of the tissue has been spared or when the dose to the salivary gland (SG) remains below a certain level. Therefore, stem cell replacement therapy may be a good option to treat radiation‐induced hyposalivation. Substantial progress has been made lately in the understanding of cell turnover in the SG, and the recent identification of stem and progenitor cell populations in the SG provides a basis for studies toward development of a stem cell‐based therapy for xerostomia. Here, we review the current state of knowledge of SG stem cells and their potential for use in a cell‐based therapy that may provide a more durable cure for hyposalivation. STEM CELLS 2013;31:613–619

Purification and Ex Vivo Expansion of Fully Functional Salivary Gland Stem Cells

Summary Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24hi/CD29hi). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells

Hematopoietic stem/progenitor cell (HSPC) traits differ between genetically distinct mouse strains. For example, DBA/2 mice have a higher HSPC frequency compared with C57BL/6 mice. We performed a genetic screen for micro-RNAs that are differentially expressed between LSK, LS(-)K(+), erythroid and myeloid cells isolated from C57BL/6 and DBA/2 mice. This analysis identified 131 micro-RNAs that were differentially expressed between cell types and 15 that were differentially expressed between mouse strains. Of special interest was an evolutionary conserved miR cluster located on chromosome 17 consisting of miR-99b, let-7e, and miR-125a. All cluster members were most highly expressed in LSKs and down-regulated upon differentiation. In addition, these microRNAs were higher expressed in DBA/2 cells compared with C57BL/6 cells, and thus correlated with HSPC frequency. To functionally characterize these microRNAs, we overexpressed the entire miR-cluster 99b/let-7e/125a and miR-125a alone in BM cells from C57BL/6 mice. Overexpression of the miR-cluster or miR-125a dramatically increased day-35 CAFC activity and caused severe hematopoietic phenotypes upon transplantation. We showed that a single member of the miR-cluster, namely miR-125a, is responsible for the majority of the observed miR-cluster overexpression effects. Finally, we performed genome-wide gene expression arrays and identified candidate target genes through which miR-125a may modulate HSPC fate.

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.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.