The dual face of parathyroid hormone and prostaglandins in the osteoimmune system

The microenvironment of bone marrow, an extraordinarily heterogeneous and dynamic system, is populated by bone and immune cells, and its functional dimension has been at the forefront of recent studies in the field of osteoimmunology. The interaction of both marrow niches supports self-renewal, differentiation, and homing of the hematopoietic stem cells and provides the essential regulatory molecules for osteoblast and osteoclast homeostasis. Impaired signaling within the niches results in a pathological tableau and enhances disease, including osteoporosis and arthritis, or the rejection of hematopoietic stem cell transplants. Discovering the anabolic players that control these mechanisms has become warranted. In this review, we focus on parathyroid hormone (PTH) and prostaglandins (PGs), potent molecular mediators, both of which carry out a multitude of functions, particularly in bone lining cells and T cells. These two regulators proved to be promising therapeutic agents when strictly clinical protocols on dose treatments were applied.

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The Role of the Bone Marrow Microenvironment in the Response to Infection

Frontiers in Immunology ◽

10.3389/fimmu.2020.585402 ◽

2020 ◽

Vol 11 ◽

Author(s):

Courtney B. Johnson ◽

Jizhou Zhang ◽

Daniel Lucas

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Immune Cells ◽

Critical Role ◽

Primary Source ◽

Bone Marrow Microenvironment ◽

Future Research ◽

Multipotent Stem Cells ◽

Hematopoietic Stem

Hematopoiesis in the bone marrow (BM) is the primary source of immune cells. Hematopoiesis is regulated by a diverse cellular microenvironment that supports stepwise differentiation of multipotent stem cells and progenitors into mature blood cells. Blood cell production is not static and the bone marrow has evolved to sense and respond to infection by rapidly generating immune cells that are quickly released into the circulation to replenish those that are consumed in the periphery. Unfortunately, infection also has deleterious effects injuring hematopoietic stem cells (HSC), inefficient hematopoiesis, and remodeling and destruction of the microenvironment. Despite its central role in immunity, the role of the microenvironment in the response to infection has not been systematically investigated. Here we summarize the key experimental evidence demonstrating a critical role of the bone marrow microenvironment in orchestrating the bone marrow response to infection and discuss areas of future research.

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The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Cells Tissues Organs ◽

10.1159/000510774 ◽

2020 ◽

pp. 1-6

Author(s):

Rebar N. Mohammed

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Absolute Number ◽

Cell Number ◽

Hematopoietic Stem ◽

The Absolute ◽

And Gender ◽

The Impact

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

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Pleiotrophin Regulates the Retention and Self-Renewal of Hematopoietic Stem Cells in the Bone Marrow Vascular Niche

Cell Reports ◽

10.1016/j.celrep.2012.09.002 ◽

2012 ◽

Vol 2 (4) ◽

pp. 964-975 ◽

Cited By ~ 80

Author(s):

Heather A. Himburg ◽

Jeffrey R. Harris ◽

Takahiro Ito ◽

Pamela Daher ◽

J. Lauren Russell ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Vascular Niche ◽

Bone Marrow Vascular Niche

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Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

Stem Cells International ◽

10.1155/2020/8885154 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Huihong Zeng ◽

Jiaoqi Cheng ◽

Ying Fan ◽

Yingying Luan ◽

Juan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽

10.1182/blood-2014-06-582924 ◽

2015 ◽

Vol 125 (17) ◽

pp. 2678-2688 ◽

Cited By ~ 64

Author(s):

Marisa Bowers ◽

Bin Zhang ◽

Yinwei Ho ◽

Puneet Agarwal ◽

Ching-Cheng Chen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Key Points ◽

Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

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Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Blood ◽

10.1182/blood-2019-127226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2460-2460 ◽

Cited By ~ 1

Author(s):

Hairui Su ◽

Szu-Mam Liu ◽

Chiao-Wang Sun ◽

Mark T. Bedford ◽

Xinyang Zhao

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Arginine Methylation ◽

Poly I:C ◽

Differentiation Potential ◽

Self Renewal ◽

Arginine Methyltransferase ◽

Hematopoietic Stem

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

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Mesenchymal Stem Cells Self-Regulate Their Differentiation To Maintain the Bone Marrow Stromal System.

Blood ◽

10.1182/blood.v104.11.2335.2335 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2335-2335

Author(s):

Iekuni Oh ◽

Akira Miyazato ◽

Hiroyuki Mano ◽

Tadashi Nagai ◽

Kazuo Muroi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cell Lines ◽

Molecular Mechanisms ◽

Adipocyte Differentiation ◽

Expression Profiles ◽

Self Renewal ◽

Hematopoietic Stem ◽

Steady Level

Abstract Mesenchymal stem cells (MSCs) account for a very small population in bone marrow stroma as a non-hematopoietic component with multipotency of differentiation into adipocytes, osteocytes and chondrocytes. MSC-derived cells are known to have hematopoiesis-supporting and immunomodulatory abilities. Although clinical applications of MSCs have already been conducted for the suppression of graft versus host disease in allogeneic stem cell transplantation and for tissue regeneration, underlying mechanisms of the biological events are still obscure. Previously, we established a differentiation model of MSCs using a mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2) (Nishikawa M et al: Blood81:1184–1192, 1993). Preadipocyte (A54) and myoblast (M1601) cell lines were cloned by treatment with 5-azacytidine. A54 cells and M1601 cells can terminally differentiate into adipocytes and myotubes, respectively, under appropriate conditions, while parent 10T1/2 cells remain undifferentiated. Moreover, A54 cells show a higher ability to support hematopoiesis compared with the other cell lines. In this study, we analyzed gene expression profiles of the three cell lines by using DNA microarray and real-time PCR to investigate molecular mechanisms for maintaining immaturity of parent 10T1/2 cells. In A54 cells, 202 genes were up-regulated, including those encoding critical factors for hematopoiesis such as SCF, Angiopoietin-1, and SDF-1 as well as genes known to be involved in adipocyte differentiation such as C/EBPα, C/EBPδ and PPAR-γ genes. These data are consistent with the hematopoiesis-supporting ability of A54 cells. During adipocyte differentiation, SCF and SDF-1 expression levels decreased in A54 cells while C/EBPα expression showed a steady level. Recently, osteoblasts have been reported to play crucial roles in “niche” for self-renewal of hematopoietic stem cells. Our results also implicate that precursor cells of non-hematopoietic components may have important roles for hematopoiesis in bone marrow. Meanwhile, in parent 10T1/2 cells, 105 genes were up-regulated, including CD90, Dlk, Wnt5α and many functionally unknown genes. Although C/EBPα expression was induced in 10T1/2 cells without differentiation under the adipocyte differentiation conditions, CD90 expression decreased, Dlk showed a steady level and Wnt5α was up-regulated. Assuming that some regulatory mechanisms are needed to keep an immature state of parent 10T1/2 cells even under the differentiation-inducible conditions, we performed following experiments. First, enforced Dlk expression in A54 cells did not inhibit terminal differentiation to adipocytes under the differentiation conditions. Second, when we cultured A54 cells in the conditioned media of parent 10T1/2 cells under the differentiation-inducible conditions, adipocyte differentiation was inhibited, suggesting that 10T1/2 cells produce some soluble molecules that can inhibit adipocyte differentiation. Since Wnt family is known to be involved in the regulation of self-renewal of several stem cells, Wnt5α may be one candidate for maintenance of “stemness” of MSCs. Taken together, the data of 10T1/2 cells suggest that MSCs can self-regulate their differentiation in the bone marrow stromal system. This concept may be important to investigate the fatty change of bone marrow in aging and in aplastic anemia.

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Immune Suppressor Factor Confers Enhanced Supporting Activity for Hematopoietic Stem Cells in Bone Marrow Stroma.

Blood ◽

10.1182/blood.v104.11.509.509 ◽

2004 ◽

Vol 104 (11) ◽

pp. 509-509

Author(s):

Hideaki Nakajima ◽

Fumi Shibata ◽

Yumi Fukuchi ◽

Yuko Goto-Koshino ◽

Miyuki Ito ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Line ◽

Proton Pump ◽

Stromal Cell ◽

Vacuolar Atpase ◽

Suppressor Factor ◽

Self Renewal ◽

Hematopoietic Stem ◽

Immune Suppressor

Abstract Immune suppressor factor (ISF) is a subunit of vacuolar ATPase proton pump that plays a critical role for proton transfer to extracellular space or subcellular organs. Physiological importance of vacuolar ATPase was evident in a variety of cellular functions such as re-absorption of bones by osteoclasts, acidification of lysosomes in macrophages, and acidification of urine in kidney. However, a role for vacuolar ATPase in hematopoiesis is still unknown. We have previously identified a short form of ISF (ShIF) as a stromal cell derived factor that supports factor-independent growth of a mutant subline of Ba/F3, an IL-3 dependent murine hematipoietic cell line. In this study, we addressed whether ISF supports self-renewal and expansion of primary hematopoietic stem cells (HSC). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) enhanced their colony-forming activity as revealed by the numbers and the size of the colonies. In addition, the numbers of long-term culture initiating cell (LTC-IC) were markedly increased by co-culture on MS10/ISF or MS10/ShIF. Moreover, competitive repopulating activity of c-Kit+Sca-1+Lin- HSC was significantly maintained without any added cytokines when they were co-cultured with MS10/ISF or MS10/ShIF compared with a mock control. In order to check whether these activities were dependent on the proton pump function of ISF, we generated a mutant ISF or ShIF whose proton pump activity was abolished by point mutation. Interestingly, all stem cell supporting activities described above were abolished in the mutant ISF or ShIF, indicating that proton transfer across cellular or endosomal membrane was critical. Analysis of gene expression profile of mock and ISF-transfected cell lines revealed that any cytokines or growth factors previously known to affect hematopoiesis are not modulated at mRNA level. However, downregulation of secreted frizzled related protein (sFRP)-1, an antagonist for Wnt, and up-regulation of matrix metalloproteinase-3 (MMP-3) were clearly noted in ISF/ ShIF-overexpressing cell line, suggesting that relative increase of Wnt activity and the modulation of extracellular matrix are the key molecular events underlying the enhanced supporting activity for HSC. These results propose a novel role for ISF in self-renewal and expansion of HSC in vivo.

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Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽

10.1182/blood.v116.21.405.405 ◽

2010 ◽

Vol 116 (21) ◽

pp. 405-405

Author(s):

Kenichi Miharada ◽

Göran Karlsson ◽

Jonas Larsson ◽

Emma Larsson ◽

Kavitha Siva ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Distinct Population ◽

Self Renewal ◽

Hematopoietic Stem ◽

Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

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Stress-Induced Activation of Dormant Hematopoietic Stem Cells In Vivo,

Blood ◽

10.1182/blood.v118.21.3390.3390 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3390-3390

Author(s):

Marieke Essers ◽

Raphael Lutz ◽

Stefanie Thamm ◽

Hannah Uckelmann ◽

Stephan Wurzer ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Viral Infection ◽

Cdna Array ◽

Common Mechanism ◽

Strong Increase ◽

Self Renewal ◽

Hematopoietic Stem ◽

General Role

Abstract Abstract 3390 Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs), which are characterized by pluripotency and life long self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain quiescent and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines, as well as interactions of HSCs with its niche environment. We have recently shown that the cytokine IFNa very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNa HSCs exit G0 and enter the active cell cycle. In general, IFNa is produced in response to viral infections by cells of the immune system, and plays an important role in the host defense against the viral infection. We now questioned whether endogenous IFNa is also produced in response to other forms of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNa production in the bone marrow in vivo, we have generated MxCre; ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNa in HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in proliferation of HSCs. In contrast to the direct effect of IFNa on HSCs, in vivo and in vitro experiments have shown that the LPS induced activation of HSCs is triggered via an indirect effect of LPS on CD11b+ cells in the bone marrow. Activation of these cells via TLR4 signaling then results in increased proliferation of the HSCs, a mechanism we are currently investigating in more detail. Interestingly, LPS induced activation correlated with increased expression of Sca-1 on HSCs, similar to the increased Sca-1 expression upon IFNa treatment. As for IFNa, the upregulation of Sca-1 is required for LPS induced proliferation, since Sca-1−/− mice do not respond to LPS stimulation. Furthermore, cDNA array comparisons between HSCs treated with IFNa or LPS suggest a more common mechanism of activation, independent of the source leading to the activation. In summary, these data suggest that in addition to viral infection also other forms of bone marrow stress, like LPS, result in activation of quiescent HSCs in the bone marrow, probably via similar mechanisms. Furthermore, both IFNa and LPS induced activation of HSCs are dependent on the up-regulation of Sca-1, suggesting a more general role for Sca-1 in the activation of stem cells. Disclosures: No relevant conflicts of interest to declare.

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