Mouse Sca-1 Research Articles

CAG repeat-selective compounds reduce abundance of expanded CAG RNAs in patient cell and murine models of SCAs.

Spinocerebellar ataxias (SCAs) are a genetically heterogenous group of devastating neurodegenerative conditions for which clinical care currently focuses on managing symptoms. Across these diseases there is an unmet need for therapies that address underlying disease mechanisms. We utilised the shared CAG repeat expansion mutation causative for a large subgroup of SCAs, to develop a novel disease-gene independent and mechanism agnostic small molecule screening approach to identify compounds with therapeutic potential across multiple SCAs. Using this approach, we identified the FDA approved microtubule inhibitor Colchicine and a novel CAG-repeat binding compound that reduce expression of disease associated transcripts across SCA1, 3 and 7 patient derived fibroblast lines and the Atxn1 154Q/2Q SCA1 mouse model in a repeat selective manner. Furthermore, our lead candidate rescues dysregulated alternative splicing in Atxn1 154Q/2Q mice. This work provides the first example of small molecules capable of targeting the underlying mechanism of disease across multiple CAG SCAs.

Reactive Bergmann glia play a central role in spinocerebellar ataxia inflammation via the JNK pathway

The spinocerebellar ataxias (SCAs) are devastating neurological diseases characterized by progressive cerebellar incoordination. While neurons bear the brunt of the pathology, a growing body of evidence suggests that glial cells are also affected. It has, however, been difficult to understand the role of glia, given the diversity of subtypes, each with their individual contributions to neuronal health. Using human SCA autopsy samples we have discovered that Bergmann glia—the radial glia of the cerebellum, which form intimate functional connections with cerebellar Purkinje neurons—display inflammatory JNK-dependent c-Jun phosphorylation. This phosphorylation defines a signaling pathway not observed in other activated glial populations, providing an opportunity to isolate the role of Bergmann glia in SCA inflammation. Turning to an SCA1 mouse model as a paradigmatic SCA, we demonstrate that inhibiting the JNK pathway reduces Bergmann glia inflammation accompanied by improvements in the SCA1 phenotype both behaviorally and pathologically. These findings demonstrate the causal role for Bergmann glia inflammation in SCA1 and point to a novel therapeutic strategy that could span several ataxic syndromes where Bergmann glia inflammation is a major feature.

Cholecystokinin 1 receptor activation restores normal mTORC1 signaling and is protective to Purkinje cells of SCA mice.

SUMMARYSpinocerebellar ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia and neurodegeneration, often in cerebellar Purkinje neurons. A SCA1 mouse model, Pcp2-ATXN1[30Q]D776, has severe ataxia in absence of progressive Purkinje neuron degeneration and death. Previous RNA-seq analyses identify cerebellar upregulation of the peptide hormone cholecystokinin (Cck) in Pcp2-ATXN1[30Q]D776 mice. Importantly, absence of Cck1 receptor (Cck1R) in Pcp2-ATXN1[30Q]D776 mice confers a progressive disease with Purkinje neuron death. Administration of a Cck1R agonist, A71623, to Pcp2-ATXN1[30Q] D776;Cck−/− and Pcp2-AXTN1[82Q] mice dampens Purkinje neuron pathology and associated deficits in motor performance. In addition, A71623 administration improves motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Moreover, the Cck1R agonist A71623 corrects mTORC1 signaling and improves expression of calbindin in cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results indicate that manipulation of the Cck-Cck1R pathway is a potential therapeutic target for treatment of diseases involving Purkinje neuron degeneration.

Cytokine- and TCR-Mediated Regulation of T Cell Expression of Ly6C and Sca-1.

Ly6C and Sca-1 (Ly6A/E) are Ly6 family GPI-anchored surface molecules that are differentially expressed by multiple immune populations. Ly6C expression has been used to distinguish short-lived effector CD4+ T cells from memory precursor effector cells, whereas Sca-1 has been used in the identification of CD8+ memory stem cells. This study examines the expression patterns of these molecules and establishes that, in vitro, IL-27, type I IFN, and IFN-γ are potent inducers of Ly6C and Sca-1 in naive mouse CD4+ and CD8+ T cells, whereas TGF-β limits their expression. The induction of Ly6C and Sca-1 by IL-27 and IFN-γ is dependent on STAT1, but not STAT3 or T-bet. In mouse splenocytes, at homeostasis, Ly6C and Sca-1 expression was not restricted to effector cells, but was also found at various levels on naive and memory populations. However, in response to infection with Toxoplasma gondii, pathogen-specific T cells expressed high levels of these molecules and in this context, endogenous IL-27 and IFN-γ were required for the expression of Ly6C but not Sca-1. Together, these findings highlight the TCR-dependent and cytokine-mediated signals that modulate T cell expression of Ly6C and Sca-1 in vitro and in vivo during infection.

Abstract A003: Developing an AML mouse syngeneic model for combinatory chemotherapy and immunotherapy

Abstract Little progress has been made in treatment of acute myeloid leukemia (AML) for years. Following the success of immune checkpoint inhibitors in solid tumors, immunotherapy also offers huge promise in many hematologic malignancies, including AML. Anti-PD1 antibody is currently being investigated in combination with azacitidine in patients with relapsed AML. Other emerging immuno-oncology (I/O) therapeutics are also at different stages of preclinical research. However, we are lacking relevant animal models with intact immune system for the preclinical in vivo pharmacologic studies and mechanism-of-action analysis of I/O agents. To meet this need, we set out to develop a series of transplantable mouse AML models carrying Nras mutations and/or a set of clinical relevant fusion proteins, like AML1/ETO. Using a similar approach reported by Scott Lowe and colleagues (1), we constructed retroviral vectors expressing human AML1/ETO9a IRES GFP and NrasG12D, generated retroviruses, co-transduced the fetal liver cells, and performed syngeneic intravenous injections in C57BL/6 mice. The mice developed AML in the following months as the GFP-positive cells enriched as high as around 90% in PBMC. Leukemia cells also dominatde the spleen and bone marrow in moribund mice. The splenocytes from moribund AML1/ETO9a mice were banked and transplanted into new C57BL/6 mice for several rounds. The later passages were more malignant than the early passages. To validate the immune profiling of this model, FACS analysis was applied using mouse PD-L1, Gr-1, CD45, Mac-1, c-kit, and sca-1 antibodies for GFP-positive cells. In PBMC of moribund AML1/ETO9a mice, PD-L1 and c-kit were highly expressed, while the expression levels of Gr-1, CD45, Mac-1, and sca-1 were low. For efficacy test, we performed standard-of-care (SOC) assay with cytarabine, doxorubicin, sorafenib, and anti-PD1 antibody. The dosing started around 10 days post inoculation of 1 x 106 leukemia cells, when GFP-positive cells were 2-10%. While doxorubicin showed good response, cytarabine and sorafenib resulted in modest tumor growth inhibition, and anti-PD1 antibody failed to show any meaningful efficacy. We are currently testing more combinatory treatments, including anti-TIM3 and anti-CTLA4 antibodies. Taken together, we have developed a mouse AML model with clinical relevant mutations for preclinical efficacy assessment of combinatory chemotherapies, target therapies, and immunotherapies. Reference: 1. Zuber J, Radtke I, Pardee TS, et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev 2009. Citation Format: Xin Tang, Lily Tong, Annie Xiaoyu An, Likun Zhang, Jie Cai, Qian Shi, Jean Pierre Wery, Davy Xuesong Ouyang. Developing an AML mouse syngeneic model for combinatory chemotherapy and immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A003.

Adventitial SCA-1+ Progenitor Cell Gene Sequencing Reveals the Mechanisms of Cell Migration in Response to Hyperlipidemia.

SummaryAdventitial progenitor cells, including SCA-1+ and mesenchymal stem cells, are believed to be important in vascular remodeling. It has been shown that SCA-1+ progenitor cells are involved in neointimal hyperplasia of vein grafts, but little is known concerning their involvement in hyperlipidemia-induced atherosclerosis. We employed single-cell sequencing technology on primary adventitial mouse SCA-1+ cells from wild-type and atherosclerotic-prone (ApoE-deficient) mice and found that a group of genes controlling cell migration and matrix protein degradation was highly altered. Adventitial progenitors from ApoE-deficient mice displayed an augmented migratory potential both in vitro and in vivo. This increased migratory ability was mimicked by lipid loading to SCA-1+ cells. Furthermore, we show that lipid loading increased miRNA-29b expression and induced sirtuin-1 and matrix metalloproteinase-9 levels to promote cell migration. These results provide direct evidence that blood cholesterol levels influence vascular progenitor cell function, which could be a potential target cell for treatment of vascular disease.

Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1.

The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1+cKit+ and human CD34+ HSPC-derived CD7+ progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3+ T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)—a co-activator not only for serum response factor, but also for Gata4 and Tbx5—is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

High-throughput screening identifies compounds that enhance lentiviral transduction.

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

Differentiation and characterization of myeloid cells.

Ex vivo differentiation of myeloid cells begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34(+) antigen (human) or Sca-1(+) (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid-induced myeloid development; however, both cell lines exhibit defects in the up-regulation of late-expressed neutrophil-specific genes. Multiple murine factor-dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation; EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid; and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradiol in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR.

Pluripotent Stem Cells Derived From Mouse and Human White Mature Adipocytes

White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

Pharmacometabolomic Signature of Ataxia SCA1 Mouse Model and Lithium Effects

We have shown that lithium treatment improves motor coordination in a spinocerebellar ataxia type 1 (SCA1) disease mouse model (Sca1154Q/+). To learn more about disease pathogenesis and molecular contributions to the neuroprotective effects of lithium, we investigated metabolomic profiles of cerebellar tissue and plasma from SCA1-model treated and untreated mice. Metabolomic analyses of wild-type and Sca1154Q/+ mice, with and without lithium treatment, were performed using gas chromatography time-of-flight mass spectrometry and BinBase mass spectral annotations. We detected 416 metabolites, of which 130 were identified. We observed specific metabolic perturbations in Sca1154Q/+ mice and major effects of lithium on metabolism, centrally and peripherally. Compared to wild-type, Sca1154Q/+ cerebella metabolic profile revealed changes in glucose, lipids, and metabolites of the tricarboxylic acid cycle and purines. Fewer metabolic differences were noted in Sca1154Q/+ mouse plasma versus wild-type. In both genotypes, the major lithium responses in cerebellum involved energy metabolism, purines, unsaturated free fatty acids, and aromatic and sulphur-containing amino acids. The largest metabolic difference with lithium was a 10-fold increase in ascorbate levels in wild-type cerebella (p<0.002), with lower threonate levels, a major ascorbate catabolite. In contrast, Sca1154Q/+ mice that received lithium showed no elevated cerebellar ascorbate levels. Our data emphasize that lithium regulates a variety of metabolic pathways, including purine, oxidative stress and energy production pathways. The purine metabolite level, reduced in the Sca1154Q/+ mice and restored upon lithium treatment, might relate to lithium neuroprotective properties.

Abstract 250: Adult Cardiac Stem Cells regulation by microRNAs

In recent years stem and progenitor cells have been identified in the heart, changing our understanding and therapeutic approach of heart disease. These cells can proliferate and differentiate in vitro into beating cardiomyocytes, endothelial and vascular smooth muscle cells, providing the potential for tissue regeneration and repair. microRNAs (miRs) have been identified as master switches controlling proliferation and differentiation, and in particular as key regulators of stem cells and cardiac development and function. Modulation of miR-regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnological and therapeutic applications. We have characterized the expression profile of a subset of 100 miRs with reported functions in stem cell and tissue differentiation in mouse Sca1+ cardiac progenitor cells (CPCs). CPC miR expression profiles were compared with bone marrow mesenchymal stem cells (MSCs) and mouse embryonic heart (E=9) to obtain insights into the origins and function of this rare cell population. Within the miR panel that was studied, a subset with relative high expression levels in CPCs was identified, which may act as negative regulators of differentiation and proliferation. Although the overall expression profile of Sca1+ CPCs is closer to BM-MSCs, the most highly expressed miRs in CPCs are distinctive and predicted to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. Our results provide novel insights into the gene expression networks active in CPCs, which will now be explored in functional studies to identify key regulators of proliferation and differentiation.

Oxidized low‐density lipoprotein induces hematopoietic stem cell senescence

We have investigated oxidized low-density lipoprotein (ox-LDL) induced senescence in hematopoietic stem cells (HCs). Mouse Sca-1+ HCs were separated and purified using the magnetic activated cell sorting technique. Ox-LDL induced significant senescence in HCs measured by SA-β-Gal staining, and reduced CFU-Mix colony-forming capacity, arresting cells at G0/G1 phase. In agreement with the cell cycle arrest, ox-LDL markedly reduced the expression of CDK4, cyclin D, and cyclin E. As possible contributing factors for cell senescence, ox-LDL also induced cellular oxidative stress and reduced telomerase activity.

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1+ cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.

Regulator of G‐protein signaling 18 controls megakaryopoiesis and the cilia‐mediated vertebrate mechanosensory system

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.

Xpa deficiency reduces CAG trinucleotide repeat instability in neuronal tissues in a mouse model of SCA1

Expansion of trinucleotide repeats (TNRs) is responsible for a number of human neurodegenerative disorders. The molecular mechanisms that underlie TNR instability in humans are not clear. Based on results from model systems, several mechanisms for instability have been proposed, all of which focus on the ability of TNRs to form alternative structures during normal DNA transactions, including replication, DNA repair and transcription. These abnormal structures are thought to trigger changes in TNR length. We have previously shown that transcription-induced TNR instability in cultured human cells depends on several genes known to be involved in transcription-coupled nucleotide excision repair (NER). We hypothesized that NER normally functions to destabilize expanded TNRs. To test this hypothesis, we bred an Xpa null allele, which eliminates NER, into the TNR mouse model for spinocerebellar ataxia type 1 (SCA1), which carries an expanded CAG repeat tract at the endogenous mouse Sca1 locus. We find that Xpa deficiency does not substantially affect TNR instability in either the male or female germline; however, it dramatically reduces CAG repeat instability in neuronal tissues-striatum, hippocampus and cerebral cortex-but does not alter CAG instability in kidney or liver. The tissue-specific effect of Xpa deficiency represents a novel finding; it suggests that tissue-to-tissue variation in CAG repeat instability arises, in part, by different underlying mechanisms. These results validate our original findings in cultured human cells and suggest that transcription may induce NER-dependent TNR instability in neuronal tissues in humans.

Inhibition of Sca-1-Positive Skeletal Stem Cell Recruitment by Alendronate Blunts the Anabolic Effects of Parathyroid Hormone on Bone Remodeling

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of antiresorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1(+)) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1(+) skeletal stem cells for the bone formation. The observation was validated in a Tgfb1(-/-) mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1(+) cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1(+) skeletal stem cells and impairs the anabolic action of PTH in bone.

Bone Marrow Subsets Differentiate into Endothelial Cells and Pericytes Contributing to Ewing's Tumor Vessels

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.