Congenic Mice Confirm That Collagen X Is Required for Proper Hematopoietic Development

The link between endochondral skeletal development and hematopoiesis in the marrow was established in the collagen X transgenic (Tg) and null (KO) mice. Disrupted function of collagen X, a major hypertrophic cartilage matrix protein, resulted in skeletal and hematopoietic defects in endochondrally derived tissues. Manifestation of the disease phenotype was variable, ranging from perinatal lethality in a subset of mice, to altered lymphopoiesis and impaired immunity in the surviving mice. To exclude contribution of strain specific modifiers to this variable manifestation of the skeleto-hematopoietic phenotype, C57Bl/6 and DBA/2J collagen X congenic lines were established. Comparable disease manifestations confirmed that the skeleto-hematopoietic alterations are an inherent outcome of disrupted collagen X function. Further, colony forming cell assays, complete blood count analysis, serum antibody ELISA, and organ outgrowth studies established altered lymphopoiesis in all collagen X Tg and KO mice and implicated opportunistic infection as a contributor to the severe disease phenotype. These data support a model where endochondral ossification-specific collagen X contributes to the establishment of a hematopoietic niche at the chondro-osseous junction

Genetically Engineered Triple MAPT-Mutant Human-Induced Pluripotent Stem Cells (N279K, P301L, and E10+16 Mutations) Exhibit Impairments in Mitochondrial Bioenergetics and Dynamics.

Pathological abnormalities in the tau protein give rise to a variety of neurodegenerative diseases, conjointly termed tauopathies. Several tau mutations have been identified in the tau-encoding gene MAPT, affecting either the physical properties of tau or resulting in altered tau splicing. At early disease stages, mitochondrial dysfunction was highlighted with mutant tau compromising almost every aspect of mitochondrial function. Additionally, mitochondria have emerged as fundamental regulators of stem cell function. Here, we show that compared to the isogenic wild-type triple MAPT-mutant human-induced pluripotent stem cells, bearing the pathogenic N279K, P301L, and E10+16 mutations, exhibit deficits in mitochondrial bioenergetics and present altered parameters linked to the metabolic regulation of mitochondria. Moreover, we demonstrate that the triple tau mutations disturb the cellular redox homeostasis and modify the mitochondrial network morphology and distribution. This study provides the first characterization of disease-associated tau-mediated mitochondrial impairments in an advanced human cellular tau pathology model at early disease stages, ranging from mitochondrial bioenergetics to dynamics. Consequently, comprehending better the influence of dysfunctional mitochondria on the development and differentiation of stem cells and their contribution to disease progression may thus assist in the potential prevention and treatment of tau-related neurodegeneration.Partial funding for open access charge: Universidad de Málag

Pro-Inflammatory Cytokines, IFNγ and TNFα, Influence Immune Properties of Human Bone Marrow and Wharton Jelly Mesenchymal Stem Cells Differentially

BACKGROUND: Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-gamma (IFNgamma) and Tumor Necrosis Factor-alpha (TNFalpha). METHODOLOGY/PRINCIPAL FINDINGS: Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNgamma or TNFalpha, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNgamma primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNgamma inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation. CONCLUSION/SIGNIFICANCE: This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation

Molecular Signatures of Quiescent, Mobilized and Leukemia-Initiating Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells

Epigenetic Induction of Definitive and Pancreatic Endoderm Cell Fate in Human Fibroblasts

Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions

The role of survivin in angiogenesis during zebrafish embryonic development

<p>Abstract</p> <p>Background</p> <p>Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish <it>survivin-1 </it>gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse <it>survivin </it>gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (Sur_UTR) and sequences flanking the initiation codon (Sur_ATG) of zebrafish <it>survivin-1 </it>gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in <it>Tg(fli1:EGFP)</it>^<it>y</it>1embryos. Results: In embryos co-injected with Sur_UTRand Sur_ATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of Sur_UTRand Sur_ATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of <it>survivin </it>gene and rescued by <it>survivin </it>mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.</p

Sox2 Is Required for Maintenance and Differentiation of Bronchiolar Clara, Ciliated, and Goblet Cells

The bronchioles of the murine lung are lined by a simple columnar epithelium composed of ciliated, Clara, and goblet cells that together mediate barrier function, mucociliary clearance and innate host defense, vital for pulmonary homeostasis. In the present work, we demonstrate that expression of Sox2 in Clara cells is required for the differentiation of ciliated, Clara, and goblet cells that line the bronchioles of the postnatal lung. The gene was selectively deleted in Clara cells utilizing Scgb1a1-Cre, causing the progressive loss of Sox2 in the bronchioles during perinatal and postnatal development. The rate of bronchiolar cell proliferation was decreased and associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of Clara cells (Scgb1a1), ciliated cells (FoxJ1 and α-tubulin), and goblet cells (Spdef and Muc5AC). By adulthood, bronchiolar cell numbers were decreased and Sox2 was absent in extensive regions of the bronchiolar epithelium, at which time residual Sox2 expression was primarily restricted to selective niches of CGRP staining neuroepithelial cells. Allergen-induced goblet cell differentiation and mucus production was absent in the respiratory epithelium lacking Sox2. In vitro, Sox2 activated promoter-luciferase reporter constructs for differentiation markers characteristic of Clara, ciliated, and goblet cells, Scgb1a1, FoxJ1, and Agr2, respectively. Sox2 physically interacted with Smad3 and inhibited TGF-β1/Smad3-mediated transcriptional activity in vitro, a pathway that negatively regulates proliferation. Sox2 is required for proliferation and differentiation of Clara cells that serve as the progenitor cells from which Clara, ciliated, and goblet cells are derived

Methotrexate exacerbates tumor progression in a murine model of chronic myeloid leukemia

ABSTRACT Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). 32Dp210 tumor cells were found to be sensitive to methotrexate (MTX) in vitro, whereas cells expressing the tyrosine-22 DHFR gene were protected from MTX at up to micromolar concentrations. MTX administered at low dose (2 mg/kg/day) did not protect normal C3H-He/J mice from 32Dp210 tumor infused intravenously, with drug toxicity limiting the administration of higher doses. Animals engrafted with transgenic tyrosine-22 DHFR marrow were protected from greater MTX doses (up to 6 mg/kg/day). However, the increased doses of MTX afforded by drug-resistance gene expression surprisingly resulted in decreased survival of the transplanted tumor-bearing animals, with increased levels of tumor detected in peripheral blood. This apparent exacerbation of tumor progression by MTX was not observed in DHFR transgenic mice in which all cells and tissues contain the drugresistance gene. This suggests that increased tumor progression in MTX-administered animals resulted from MTX sensitivity of a nonhematopoietic host component, thus allowing tumor expansion. We conclude that MTX exacerbates tumor progression in the 32Dp210 model of CML, and that based on this model alternate DHFR inhibitors combined with drug-resistant DHFR or other chemotherapeutic agent/drug-resistance gene combinations may be required for the application of drugresistance gene expression to the treatment of CML