Murine Cell Populations Research Articles

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

BackgroundHematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and MethodsTo address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. ResultsWe found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. ConclusionsOur data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories.▪ Disclosures:No relevant conflicts of interest to declare.

RETRACTED ARTICLE: Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors

BackgroundDNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation.CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners.In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors.MethodsTo achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses.ResultsThe analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased.ConclusionsTherefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

368. Efficacy of Various Populations of Murine Mesenchymal Stem Cells for the Treatment of Critical Limb Ischemia

368. Efficacy of Various Populations of Murine Mesenchymal Stem Cells for the Treatment of Critical Limb Ischemia

Notch Signaling is Associated with ALDH Activity and an Aggressive Metastatic Phenotype in Murine Osteosarcoma Cells

Osteosarcoma (OS) is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4) are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH) activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis.

Identification, characterization, and isolation of a common progenitor for osteoclasts, macrophages, and dendritic cells from murine bone marrow and periphery

Osteoclasts are specialized bone-resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype B220(-) CD3(-) CD11b(-/low) CD115(+) and either CD117(hi), CD117(intermediate), or CD117(low). We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single-cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages, and antigen-presenting dendritic cells in vitro with efficiencies of more than 90%, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages, and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined.

A Cell-Autonomous Myeloproliferative Phenotype Caused by Loss of Latexin in Stem and Progenitor Cells.

Abstract 2314We have previously shown a novel role for Latexin (Lxn), a carboxypeptidase inhibitor, in influencing the size of murine hematopoietic stem cell (HSC) population. Lxn negatively regulates HSC population size by enhancing apoptosis while repressing proliferation and self-renewal. However, the major mechanisms underlying this phenotype remain unknown. To further study the functionality of latexin in hematopoiesis, we generated a knock-out mouse model (Lxn−/− mouse) with a truncation of Lxn exon 2 to exon 5.Blood cell counts showed that lymphocytes, neutrophils, eosinophils, and monocytes were significantly increased from 50% to 100%, indicating an overall myeloid expansion in the periphery. The Lxn−/− mice also showed hypercellular bone marrow (BM), splenomegaly, and increased colony formation in methylcellulose.We found that the frequencies and absolute numbers of Lineage-/Sca-1+/c-Kit+ (LSK) and multi-potent progenitor (MPP) cells were significantly increased, while short-term and long-term HSCs remained unchanged in Lxn−/− mice. We next evaluated both lymphoid and myeloid progenitor subpopulations within the MPP compartment. Absolute numbers of granulocyte-monocyte progenitor (GMP) cells as well as common lymphoid progenitor (CLP) cells were significantly increased by 60–80% in Lxn−/− mice compared with wild type animals.We then performed cobblestone-area-forming-cell (CAFC) assay, an in vitro functional assay to evaluate the numbers of functional progenitors and stem cells. As a result, cobblestone area forming cells were significantly up-regulated at days 7, 14, 28 and 35, indicating that numbers of both mature progenitors and functional stem cells in BM were increased.To further investigate the observed progenitor and stem cell expansion in Lxn−/− mice, we studied the apoptotic status of BM cells, using the apoptotic marker Annexin V. The apoptosis frequencies were reduced approximately 50% in Lxn deficient LSK cells and MPP cells, suggesting that Lxn positively regulates apoptosis apoptosis in HSCs, as reported previously.Moreover, we used the competitive repopulation assay to determine functional capacities of Lxn−/− HSCs. In this assay, equal numbers (1×106) of Lxn−/− (or wild type) whole BM cells were transplanted along with competitors into lethally irradiated recipient animals. We analyzed the peripheral blood and BM of recipients at 15 weeks post transplantation. In both BM and PB, Lxn−/− cells showed a 50% increase in engraftment. We noted that there was significant up-regulation in granulocytes and monocytes (50%), as well as B cell lymphocytes (50%), but not in T cells, which is consistent with what we observed in Lxn−/− mice. These findings reveal that the myeloproliferation in Lxn−/− mice is transplantable and thus is intrinsic.To gain insights into the molecular mechanisms underlying the phenotypes created by loss of Lxn expression, we carried out gene profiling between Lxn and wild type MPP cells. Microarray experiments were performed on BM cells using Affymetrix Gene Chip. To study the pathway changes in response to loss of Lxn expression, we performed the gene set enrichment analysis (GSEA). Five enriched pathways have been identified as altered in Lxn−/− MPP cells that include: cell communication, ECM receptor interaction, proteasome, cell cycle and neuroactive ligand receptor interaction.In conclusion, these data showed that loss of Lxn leads to proliferative hematopoiesis with clonal expansion at the stem and progenitor cell levels, and that this myeloproliferative phenotype is cell autonomous and transplantable.This work was supported by NIH HD-060915 and the Edward P. Evans Foundation Disclosures:No relevant conflicts of interest to declare.

The role of 1α,25‐dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL‐10+CD4+T cells

1α,25-Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL-10-secreting CD4(+) T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3(+) regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3(+) and IL-10(+) CD4(+) T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL-10 at more moderate levels, with little coexpression of these molecules. The Foxp3(+) and IL-10(+) T-cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL-10. 1α25VitD3 enables the selective expansion of Foxp3(+) Treg cells over their Foxp3(-) T-cell counterparts. Equally, 1α25VitD3 maintains Foxp3(+) expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4(+) Foxp3(+) T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL-10(+) and Foxp3(+) Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.

Application of Fluorescent In Situ Hybridization in the Mouse Xenograft Model of Human Fat Grafting

Determining the optimal technique for autologous fat grafting requires elucidation of the engraftment process at the cellular level. The nude mouse xenograft model for autologous human fat grafting is an excellent tool to evaluate the incorporation of grafted fat into the recipient. The authors present a murine xenograft model that uses fluorescent in situ hybridization (FISH) to differentiate between murine (host) cells and human (grafted) cells. Fat grafts were harvested from human abdominoplasty specimens, fixed, embedded in paraffin blocks, and cut into 5 µm sections. The sections were stained and used for in situ hybridization with fluorescently labeled murine and human nucleic acid reagents. A fluorescence microscope was used for photographic analysis of the grafts, allowing identification of murine and human cell populations based on the wavelength at which they stained. DAPI (4,'6-diamidino-2-phenylindole) counterstaining was also applied to confirm that the images obtained represented cell nuclei. At 20× magnification, strong species-specific staining of cells was seen within the tissue sections, which allowed the authors to easily discriminate between graft-derived and host-derived cells. Using this approach, human fat xenografts were revealed to comprise a complex matrix of closely interacting graft- and host-derived structures. FISH has the potential to be a powerful technique for distinguishing between murine and human cells in the nude mouse xenograft model of human fat grafting. By applying this technique, it may be possible to evaluate the engraftment process at a cellular level, which may ultimately allow clinicians to obtain more predictable results with grafted fat.

Isolation and Characterization of Murine Mandibular Condylar Cartilage Cell Populations

Objectives: The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. Methods: Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. Results: Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. Conclusion: Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway.

Abstract SY12-01: Stem cell approaches dissect lung cancer biology

Abstract Lung cancer is the major cause of death from cancer worldwide. The five-year survival rate for the majority of lung cancer patients is only 16% because their tumors are refractory to chemotherapeutics or quickly become resistant to therapeutic response. Also contributing to morbidity, most lung cancer patients already have advanced, metastatic disease at the time of diagnosis or develop metastases after surgical removal of early-stage primary lung tumors. The cancer stem cell (CSC) hypothesis is an attractive explanation for tumor growth, recurrence after treatment, and metastasis in some cancers, yet the precise role of CSCs in lung tumors has not been established. Since successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor, it is crucial to characterize the cells that are able to propagate a tumor in vivo. We refer to these functionally important cancer cells as tumor-propagating cells (TPCs). Importantly, understanding the identity and molecular nature of TPCs will provide new opportunities for early detection, treatment and prevention of metastatic disease, whether or not they fit the CSC model in each type of cancer. My laboratory recently showed that murine lung adenocarcinomas, the most prevalent lung cancer subtype, contain a TPC population. In mice bearing tumors initiated with oncogenic Kras and deletion of p53, the cell surface marker Sca1 identifies cancer cells with stem cell properties. In contrast, TPCs are Sca1-negative in lung tumors that express a mutant EGFR. Thus, the predominant driving oncogenotype is an important determinant in the identification of TPC phenotype in lung cancer. This finding suggests that treatments directed against cancer stem cells should be targeted to subsets of patients grouped according to their oncogenotype. We have recently obtained global gene expression data from the Sca1-sorted populations of murine lung tumor cells, as well as their normal wild-type counterparts, to uncover the molecular pathways responsible for tumor propagation in lung adenocarcinoma. I will also discuss new avenues we have taken to understand the connections between TPCs and metastasis in mouse models as well as translating the importance of our findings in human lung cancer cells and patient samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY12-01. doi:10.1158/1538-7445.AM2011-SY12-01

Lentivirus-mediated Wnt11 Gene Transfer Enhances Cardiomyogenic Differentiation of Skeletal Muscle-derived Stem Cells

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Regulation of Granulocyte and Macrophage Populations of Murine Bone Marrow Cells by G-CSF and CD137 Protein

BackgroundGranulocytes and monocytes/macrophages differentiate from common myeloid progenitor cells. Granulocyte colony-stimulating factor (G-CSF) and CD137 (4-1BB, TNFRSF9) are growth and differentiation factors that induce granulocyte and macrophage survival and differentiation, respectively. This study describes the influence of G-CSF and recombinant CD137-Fc protein on myelopoiesis.Methodology/Principal FindingsBoth, G-CSF and CD137 protein support proliferation and survival of murine bone marrow cells. G-CSF enhances granulocyte numbers while CD137 protein enhances macrophage numbers. Both growth factors together give rise to more cells than each factor alone. Titration of G-CSF and CD137 protein dose-dependently changes the granulocyte/macrophage ratio in bone marrow cells. Both factors individually induce proliferation of hematopoietic progenitor cells (lin-, c-kit+) and differentiation to granulocytes and macrophages, respectively. The combination of G-CSF and CD137 protein further increases proliferation, and results in a higher number of macrophages than CD137 protein alone, and a lower number of granulocytes than G-CSF alone demonstrating that CD137 protein-induced monocytic differentiation is dominant over G-CSF-induced granulocytic differentiation. CD137 protein induces monocytic differentiation even in early hematopoietic progenitor cells, the common myeloid progenitors and the granulocyte macrophage progenitors.Conclusions/SignificanceThis study confirms earlier data on the regulation of myelopoiesis by CD137 receptor - ligand interaction, and extends them by demonstrating the restriction of this growth promoting influence to the monocytic lineage.

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

AbstractAbstract 405Cripto 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.

Abstract 3469: Effect of age and mutagen on the enriched murine mammary epithelial stem and progenitor cell population

Abstract Epidemiological studies indicated that aging is a significant risk factor for breast cancer. Yet, there is insufficient knowledge on the mechanisms affecting breast cancer initiation and promotion in older women. Recent studies indicated epithelial stem/progenitor cell population, responsible for lifelong tissue maintenance and regeneration, as a target of transformation. It has been reported earlier by serial transplantation of murine mammary tissues that functional mammary outgrowths indicating stem cell function is unaffected by the age of the donor. But it is unclear that whether spontaneous or induced mutagenesis in the aging stem/progenitor cell population has the potential to impair stem/progenitor functions and/or to initiate tumorigenesis. In this study, first we have isolated lineage negative (CD31−CD45−Ter119−) mammary epithelial cells (Lin−MEC) from the thoracic/inguinal mammary glands of young (6 months, n=7) and old (28 months, n=6) mice by serial enzymatic dissociation followed by magnetic nanoparticle separation method (StemCell Technology). These cells were utilized to form mammospheres, representing mammary stem/early progenitor cells. Although the frequency of mammosphere formation remained the same, the number of larger mammospheres (&amp;gt;60µm) from old mice were significantly lower than the young mice, probably indicating decreased stem cell activity in the older population. To study the effect of mutagen in vivo, we treated two young and two old mice with methyl nitrosourea (MNU, 25mg/kg body weight) once a week for three consecutive weeks. Animals were sacrificed one week after the last treatment. Both total and larger mammospheres (&amp;gt;60µm) from the isolated Lin−MEC of the treated older mice were observed in significantly higher number than the treated younger mice. In a recent clinical study larger mammospheres (&amp;gt;50µm) obtained from metastatic breast cancer cells has been correlated to their ability to induce tumors. These Lin−MEC cells were also analyzed and compared for DNA damage by alkaline comet assay and a higher percentage of cells (&amp;gt;10%) with irreversible DNA damage in the older population were observed compared to younger population (&amp;lt;2%) indicating probable lower DNA repair capacity of the former cells. Thus, our preliminary study indicated that enriched stem/progenitor cells in the old mice with decreased in vitro stem cell activity might have the potential to be vulnerable to DNA damage by mutagens and subsequent tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3469.

H4 Histamine Receptors Mediate Cell Cycle Arrest in Growth Factor-Induced Murine and Human Hematopoietic Progenitor Cells

The most recently characterized H4 histamine receptor (H4R) is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

Regulation of Stem Cell Aging by Retinoblastoma Like-1 (p107)

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. We have previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged C57BL/6 (B6) and DBA/2 (D2) mice. In B6 mice the HSC population steadily increases with age, whereas in D2 mice, this population declines. A QTL regulating the natural variation in lifespan between the two strains was mapped to the same location on mouse Chr 2, thus leading to the hypothesis that stem cell function affects longevity. B6 alleles of this locus, associated with expansion of the stem cell pool, are also associated with a ~50% increase in lifespan. In the present study, we characterize a congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, we analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and self-renewal capabilities of HSCs. Next, we used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Three variables were examined using Affymetrix M430 arrays:the effect of strain—congenic versus background;the effect of age—2 months versus 22 months; andthe effects of 2 Gy of irradiation because previous studies indicated that congenic animals were highly sensitive to the effects of mild radiation compared to B6 background animals.Extensive analysis of the expression arrays pointed to a strong candidate, the gene encoding Retinoblastoma like protein 1, otherwise known as p107. The B6 allele is associated with increased p107 expression in old HSCs therefore p107 in this context is a positive regulator of stem cell number in aged mice. Real-time PCR was used to validate the differential expression of p107 in lineage negative and lineage negative Sca-1+, c-kit+ (LSK) cells. Detailed sequence analysis of the gene revealed the presence of 4 non-synonymous, coding region single nucleotide polymorphisms (SNPs) between B6 and D2 mice, which may contribute to the differential expression of the gene and function of the protein. Perhaps most importantly, we show that overexpression of p107 in congenic HSCs increases day 21, day 28, and day 35 CAFC numbers in vivo by 2- to 4-fold, therefore confirming its role as a positive regulator of primitive progenitor populations including HSCs. These studies uncover a novel role for p107 and provide additional clues in the complex regulation of stem cell aging.

Comprehensive Profiling of Micrornas in Murine Hematopoietic Stem Cells and Lineages Using a Microfluidics Approach

MicroRNAs (miRNAs) have been shown to be developmental regulators in various organisms and tissues such as the hematopoietic system. miRNA profiling studies have been primarily performed on specific aspects of hematopoiesis like lymphocyte or red blood cell development. However, a comprehensive study including rare hematopoietic stem cell populations and various lineages has yet to be published. MiRNA expression profiling within the hematopoietic tree is challenging due to difficulties in obtaining highly purified samples of stem and progenitor cell populations as well as the high cost and labour associated with global profiling approaches. The combined requirements of high sensitivity, dynamic range and efficient throughput pose serious obstacles to the use of established methods including Northern Blot, cloning, miRNA microarrays, and deep sequencing. Real time PCR offers the requisite dynamic range and sensitivity, but is labour intensive and prohibitive in cost using conventional formats. To overcome these limitations we combined new high throughput microfluidic technologies with a 288-plex real time PCR approach to quantify miRNAs in hematopoietic stem cells and lineage positive cells. This approach allowed us the simultaneous detection of 288 miRNAs in small numbers (≤3000) of cells across multiple subpopulations of the murine hematopoietic tree. Twenty unique murine hematopoietic cell populations were isolated through current FACS sorting strategies, including hematopoietic stem cells (HSCs) based on SLAM and LSK markers, myeloid and lymphoid progenitor cells as well as mature populations of all lineages. The cells were immediately lysed after sorting and reverse transcribed using 3 pools of 96 stemloop RT-primers, followed by a PCR pre-amplification step. In order to detect individual amplified products, we used BioMark™ 48.48 Dynamic Arrays (Fluidigm Corp, San Francisco, USA) and miRNA specific TaqMan probes. For each miRNA, a series of synthetic miRNA dilutions was used as a standard to determine the absolute number of miRNA molecules per cell. This analysis further revealed systematic and miRNA-specific variations in the sensitivities of Taqman assays, highlighting that RT-PCR analysis without the inclusion of an absolute standard may be misrepresentative of the true molecular abundance. Hierarchical clustering analysis and comparison between hematopoietic stem cell (HSC) populations and mature populations revealed miRNAs that are critical for hematopoietic development and maturation. In general, miRNAs detected at the highest abundance were miR-706 and miR-720, which is likely due to highly reactive Taqman assays for these targets. Of the tested 288 miRNAs, only 133 were detected across all cell populations. Most of these miRNAs exhibited a mixed expression profile, with expression peaks in the differentiated populations. Consistent with previous results, we detected a strong increase of miR-223 within myeloid differentiated populations. The highest levels were detected in neutrophils and monocytes, but surprisingly low levels were found in mast cells, supporting the specific role of miR-223 in myeloid differentiation. Other miRNAs highly enriched in differentiated cells were miR-142 and miR-16. Clustering revealed a distinct miRNA expression pattern for the profiled HSC populations including miRNAs located in the Hox cluster and the miR-181 family. In conclusion, we applied a novel technical approach to quantify a broad range of miRNAs in rare cell populations. With this approach we can further define the expression patterns of miRNAs from hematopoietic stem cells to mature lineages.

Murine Hematopoietic Stem Cells and Multipotent Progenitors Express Truncated Intracellular Form of c-kit Receptor

The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors.

Proteomic Differentiation Between Murine Retinal and Brain-Derived Progenitor Cells

Previous studies have transplanted a variety of neural stem cells (NSCs) to the eye in hopes of developing a therapy to replace retinal neurons lost to disease. Successful integration, survival, and differentiation of the cell types has been variably successful. At the moment, little is known about the fundamental biological differences between stem cell or progenitor cell types. Characterization of these differences will not only increase our general understanding of this broadly characterized group of cells, but also lead to development of criteria for sorting cells, evaluating their differentiation, and predicting their suitability for transplantation. We have used two-dimensional gel electrophoresis protein expression profiles to characterize the molecular differences between two populations of murine progenitor cells-retinal progenitor cells (RPCs) and brain progenitor cells (BPCs) isolated from mice of the same age and same genetic background. Our protein expression profiling identified 22 proteins that are differentially expressed in RPCs when compared to BPCs. Four of the differentially expressed proteins correspond to proteins known to be involved in a cellular response to stress, and analysis of potential transcription factor binding sites in the promoter regions of their genes suggests these proteins could be co-regulated at the transcriptional level. On the basis of this discovery, we tested the hypothesis that the addition of the antioxidant vitamin E would decrease the expression of the stress-response proteins and influence differentiation of RPCs. Further investigation of differences between multiple populations of RPCs and BPCs during their maintenance and differentiation will further identify fundamental differences that define 'retinal-like' characteristics and provide tools to assay the success of efforts to influence many populations of stem cells to adapt a retinal cell fate.

Prion strain discrimination in cell culture: The cell panel assay

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).