Sequential <em>In vivo</em> Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

real-time-imaging-ccl5-induced-migration-periosteal-skeletal-stem

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Harvard Stem Cell Institute

Baylor College of Medicine

Current dendritic cell (DC) vaccine preparations involving ex vivo differentiation and maturation produce short-lived, transiently active DCs that may curtail T-cell responses in vivo. We demonstrate that Akt1, downregulation of which decreases DC lifespan, is critical for proinflammatory signal-mediated DC survival and maturation. Lipopolysaccharide or CD40 signaling stabilizes Akt1, promoting both activation and Bcl-2-dependent survival of DCs. Expression of a potent allele encoding a lipid raft-targeted Akt1, M(F)-DeltaAkt, is sufficient for maturation and survival of murine bone marrow-derived DCs in vivo. M(F)-DeltaAkt-transduced DCs enhanced T-cell proliferation, activation and long-term memory responses, enabling eradication of large pre-established lymphomas and aggressive B16 melanomas. Human myeloid DCs expressing constitutively active M(F)-DeltahAkt also survived significantly longer and promoted antigen-specific T-cell responses. Thus, Akt1 is a critical regulator of DC lifespan, and its manipulation in DCs can improve the clinical efficacy of DC-based tumor vaccines.

An essential role for Akt1 in dendritic cell function and tumor immunotherapy.

Lck-interacting adaptor protein/Rlk/Itk-binding protein (Lad/RIBP) was previously identified as an adaptor protein involved in TCR-mediated T-cell activation. To elucidate the functions of Lad further, we here performed yeast 2-hybrid screening using Lad as bait and discovered that the G protein beta subunit (G beta) is a Lad-binding partner. Since the most well-known G protein-coupled receptor in T cells is the chemokine receptor, we investigated whether Lad is involved in chemokine signaling. We found that, upon chemokine treatment, Lad associated with G beta in Jurkat T cells. Furthermore, ectopic expression of dominant-negative Lad or the reduction of endogenous Lad expression by siRNA impaired the chemokine-induced migration of T cells, indicating that Lad is required for chemokine-induced T-cell migration. Subsequent investigation of the signaling pathways revealed that, in response to chemokine, Lad associated with the tyrosine kinases Lck and Zap-70 and that Lad was essential for the activation of Zap-70. Moreover, Lad was required for the chemokine-dependent tyrosine phosphorylation of focal adhesion molecules that included Pyk2 and paxillin. Taken together, these data show that, upon chemokine stimulation, Lad acts as an adaptor protein that links the G protein beta subunit to the tyrosine kinases Lck and Zap-70, thereby mediating T-cell migration.

The adaptor protein Lad associates with the G protein beta subunit and mediates chemokine-dependent T-cell migration.

&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#xFF08;HSC&#xFF09;&#x306F;&#x3001;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x3057;&#x3001;&#x8907;&#x6570;&#x306E;&#x7570;&#x306A;&#x308B;&#x9020;&#x8840;&#x7CFB;&#x7D71;&#x306B;&#x6CBF;&#x3063;&#x3066;&#x6210;&#x719F;&#x3057;&#x305F;&#x5B50;&#x5B6B;&#x306B;&#x5206;&#x5316;&#x3059;&#x308B;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x6A5F;&#x80FD;&#x304C;&#x9AD8;&#x5EA6;&#x306B;&#x5C02;&#x9580;&#x7684;&#x306A;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x5185;&#x3067;&#x898B;&#x3064;&#x304B;&#x3063;&#x305F;&#x5468;&#x56F2;&#x306E;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x306E;&#x4FE1;&#x53F7;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x3068;&#x547C;&#x3070;&#x308C;&#x308B;&#x3002; HSC&#x30CB;&#x30C3;&#x30C1;&#x3092;&#x7406;&#x89E3;&#x3057;&#x3001;&#x6D3B;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x540C;&#x69D8;&#x306B;&#x57FA;&#x672C;&#x7684;&#x306A;&#x79D1;&#x5B66;&#x8005;&#x3084;&#x81E8;&#x5E8A;&#x533B;&#x306E;&#x76EE;&#x6A19;&#x3067;&#x3059;&#x3002;&#x6700;&#x8FD1;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306B;&#x91CD;&#x8981;&#x306A;&#x7D30;&#x80DE;&#x6210;&#x5206;&#x3084;&#x5206;&#x5B50;&#x306E;&#x8981;&#x56E0;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x7126;&#x70B9;&#x3092;&#x5F53;&#x3066;&#x3066;&#x3044;&#x308B;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6700;&#x8FD1;&#x306E;&#x77E5;&#x898B;&#x3092;&#x78BA;&#x8A8D;&#x3059;&#x308B;&#x672A;&#x89E3;&#x6C7A;&#x306E;&#x554F;&#x984C;&#x3092;&#x8B70;&#x8AD6;&#x3057;&#x3001;HSC&#x30CB;&#x30C3;&#x30C1;&#x306E;&#x79C1;&#x305F;&#x3061;&#x306E;&#x73FE;&#x5728;&#x306E;&#x77E5;&#x8B58;&#x306E;&#x81E8;&#x5E8A;&#x7684;&#x610F;&#x7FA9;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x3002;

&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x30CB;&#x30C3;&#x30C1;

&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE; (&#x661F;) &#x62E5;&#x6709; self-renew &#x548C;&#x533A;&#x5206;&#x5230;&#x6CBF;&#x591A;&#x4E2A;&#x4E0D;&#x540C;&#x7684;&#x9020;&#x8840;&#x8C31;&#x7CFB;&#x7684;&#x6210;&#x719F;&#x540E;&#x4EE3;&#x7684;&#x80FD;&#x529B;&#x3002;&#x661F;&#x7684;&#x529F;&#x80FD;&#x53D6;&#x51B3;&#x4E8E;&#x6765;&#x81EA;&#x5468;&#x56F4;&#x7684;&#x5355;&#x5143;&#x683C;&#xFF0C;&#x5728;&#x9AD8;&#x5EA6;&#x4E13;&#x4E1A;&#x5316;&#x7684;&#x5FAE;&#x73AF;&#x5883;&#x79F0;&#x4E3A;&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE;&#x9F9B;&#x5185;&#x53D1;&#x73B0;&#x7684;&#x4FE1;&#x53F7;&#x3002;&#x4E86;&#x89E3;&#x5E76;&#x5229;&#x7528; HSC &#x5229;&#x57FA;&#x662F;&#x4E00;&#x4E2A;&#x57FA;&#x672C;&#x7684;&#x79D1;&#x5B66;&#x5BB6;&#x548C;&#x4E34;&#x5E8A;&#x533B;&#x751F;&#x90FD;&#x7684;&#x76EE;&#x6807;&#x3002;&#x6700;&#x8FD1;&#x7684;&#x7814;&#x7A76;&#x96C6;&#x4E2D;&#x5728;&#x5BF9;&#x6B64;&#x5FAE;&#x73AF;&#x5883;&#x5B9A;&#x4E49;&#x7684;&#x7EC6;&#x80DE;&#x6210;&#x5206;&#x548C;&#x5206;&#x5B50;&#x7684;&#x5173;&#x952E;&#x56E0;&#x7D20;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x68C0;&#x8BA8;&#x6700;&#x8FD1;&#x7684;&#x8C03;&#x67E5;&#x7ED3;&#x679C;&#xFF0C;&#x8BA8;&#x8BBA;&#x5C1A;&#x672A;&#x89E3;&#x51B3;&#x7684;&#x95EE;&#x9898;&#xFF0C;&#x5E76;&#x68C0;&#x67E5;&#x6211;&#x4EEC;&#x76EE;&#x524D;&#x6240;&#x77E5;&#x7684; HSC &#x751F;&#x6001;&#x4F4D;&#x7684;&#x4E34;&#x5E8A;&#x610F;&#x4E49;&#x3002;

&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE;&#x9F9B;&#x3002;

Hematopoietic stem cells (HSCs) possess the ability to self-renew and to differentiate to mature progeny along multiple different hematopoietic lineages. The function of HSCs depends upon the signals from surrounding cells found within the highly specialized microenvironment termed the hematopoietic stem cell niche. Understanding and exploiting the HSC niche is a goal of basic scientists and clinicians alike. Recent studies have focused on defining the cellular components and molecular factors critical to this microenvironment. Here we review recent findings, discuss unresolved questions, and examine the clinical implications of our current knowledge of the HSC niche.

The hematopoietic stem cell niche.

Les cellules souches h&#xE9;matopo&#xEF;&#xE9;tiques (CSH) poss&#xE8;dent la capacit&#xE9; de s&#x26;#39;auto-renouveler et de faire la diff&#xE9;rence &#xE0; la descendance matures le long de plusieurs diff&#xE9;rentes lign&#xE9;es h&#xE9;matopo&#xEF;&#xE9;tiques. La fonction de CSH d&#xE9;pend des signaux provenant de cellules environnantes trouv&#xE9;s dans le microenvironnement hautement sp&#xE9;cialis&#xE9; appel&#xE9; la niche de cellules souches h&#xE9;matopo&#xEF;&#xE9;tiques. Comprendre et exploiter le cr&#xE9;neau HSC est un objectif des scientifiques de base et les cliniciens. Des &#xE9;tudes r&#xE9;centes ont mis l&#x26;#39;accent sur la d&#xE9;finition des composants cellulaires et mol&#xE9;culaires des facteurs critiques pour ce micro-environnement. Ici nous passons en revue des r&#xE9;sultats r&#xE9;cents, discuter de questions non r&#xE9;solues, et d&#x26;#39;examiner les implications cliniques de nos connaissances actuelles de la niche HSC.

La Niche de cellules souches h&#xE9;matopo&#xEF;&#xE9;tiques

H&auml;matopoetischen Stammzellen (HSCs) besitzen die F&auml;higkeit zu erneuern und zu Reifen Nachkommen entlang mehrere verschiedene &Uuml;bertragungslinien h&auml;matopoetischen unterscheiden. Die Funktion der HSCs h&auml;ngt die Signale vom umgebenden Zellen innerhalb der hoch spezialisierten Mikromilieu bezeichnet die h&auml;matopoetische Stammzelle Nische gefunden. Verstehen und die HSC-Nische ausbeuten ist ein Ziel von Grundlagenforschern und Klinikern gleicherma&szlig;en. J&uuml;ngste Studien konzentrierten sich auf die Definition von Zellbestandteilen und Molekulare Faktoren kritisch zu diesem Untersuchungsverfahren. Hier &uuml;berpr&uuml;fen wir neuere Befunde, offene Fragen zu diskutieren, und untersuchen Sie die klinischen Auswirkungen unseres aktuellen Wissens der HSC Nische.

Die h&auml;matopoetische Stammzelle Nische.

Cellule staminali ematopoietiche (HSCs) possiedono la capacit&agrave; di auto-rinnovarsi e di differenziare progenie matura lungo pi&ugrave; diversi lignaggi ematopoietici. La funzione di HSCs dipende i segnali dalle cellule circostanti, trovate all&#39;interno del microambiente altamente specializzato definito la nicchia delle cellule staminali ematopoietiche. Capire e sfruttare la nicchia HSC &egrave; un obiettivo di base scienziati e medici allo stesso modo. Recenti studi si sono concentrati sulla definizione delle componenti cellulari e molecolari fattori critici a questo microambiente. Qui recensiamo le recenti scoperte, discutere di questioni irrisolte ed esaminare le implicazioni cliniche delle nostre conoscenze attuali della nicchia HSC.

La nicchia delle cellule staminali ematopoietiche.

&#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; (HSCs) self-renew&#xC640; &#xC5EC;&#xB7EC; &#xB2E4;&#xB978; &#xC870; &#xD608; &#xACC4;&#xBCF4;&#xB97C; &#xB530;&#xB77C; &#xC131;&#xC219; &#xC790;&#xC190;&#xC744; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; &#xB2A5;&#xB825;&#xC744; &#xBCF4;&#xC720;. HSCs &#xAE30;&#xB2A5; &#xB418; &#xB098; &#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD2C8;&#xC0C8;&#xB294; &#xACE0;&#xB3C4;&#xB85C; &#xC804;&#xBB38;&#xD654; &#xB41C; microenvironment &#xC8FC;&#xBCC0; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC2E0;&#xD638;&#xC5D0; &#xB530;&#xB77C; &#xB2E4;&#xB985;&#xB2C8;&#xB2E4;. &#xC774;&#xD574; &#xD558; &#xACE0; &#xC545;&#xC6A9; &#xD558;&#xB294; HSC &#xD2C8;&#xC0C8; &#xAE30;&#xBCF8; &#xACFC;&#xD559;&#xC790;&#xC640; &#xC784;&#xC0C1; &#xBAA8;&#xB450;&#xC758; &#xBAA9;&#xD45C;&#xC785;&#xB2C8;&#xB2E4;. &#xCD5C;&#xADFC; &#xC5F0;&#xAD6C;&#xB294; &#xC138;&#xD3EC; &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xBC0F; &#xC911;&#xC694; &#xD55C; &#xBD84;&#xC790; &#xC694;&#xC778;&#xC774;&#xC774; microenvironment&#xC5D0; &#xC815;&#xC758;&#xC5D0; &#xC9D1;&#xC911; &#xD588;&#xB2E4;. &#xCD5C;&#xADFC; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xAC80;&#xD1A0; &#xD558;&#xB294; &#xC5EC;&#xAE30;&#xC11C; &#xD574;&#xACB0; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xC9C8;&#xBB38;&#xC744; &#xD1A0;&#xB860; &#xD558; &#xACE0; &#xC6B0;&#xB9AC;&#xC758; &#xD604;&#xC7AC;&#xC758; &#xC9C0;&#xC2DD;&#xC744; HSC &#xD2C8;&#xC0C8;&#xC758; &#xC784;&#xC0C1; &#xC758;&#xBBF8;&#xB97C; &#xAC80;&#xC0AC; &#xD569;&#xB2C8;&#xB2E4;.

&#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD2C8;&#xC0C8;&#xC785;&#xB2C8;&#xB2E4;.

Las c&#xE9;lulas madre hematopoy&#xE9;ticas (HSC), poseen la capacidad de auto-renovarse y diferenciar a la progenie madura a lo largo de varios linajes hematopoy&#xE9;ticos diferentes. La funci&#xF3;n de las CMH depende de las se&#xF1;ales de las c&#xE9;lulas circundantes se encuentran en el microambiente altamente especializada denomina el nicho de c&#xE9;lulas madre hematopoy&#xE9;ticas. Entender y explotar el nicho de HSC es un objetivo de los cient&#xED;ficos b&#xE1;sicos y cl&#xED;nicos por igual. En estudios recientes se han centrado en la definici&#xF3;n de los componentes celulares y factores moleculares cr&#xED;ticas para este microambiente. En este art&#xED;culo revisamos los hallazgos recientes, discutir las cuestiones no resueltas, y examinar las implicaciones cl&#xED;nicas de nuestro conocimiento actual del nicho de HSC.

La madre hematopoy&#xE9;ticas nicho de c&#xE9;lulas

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.

Activation of Wnt signaling by chemically induced dimerization of LRP5 disrupts cellular homeostasis.

Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.

Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.

Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.

Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89.

Analysis of biomedical images requires computational expertize that are uncommon among biomedical scientists. Deep learning approaches for image analysis provide an opportunity to develop user-friendly tools for exploratory data analysis. Here, we use the visual programming toolbox Orange ( http://orange.biolab.si ) to simplify image analysis by integrating deep-learning embedding, machine learning procedures, and data visualization. Orange supports the construction of data analysis workflows by assembling components for data preprocessing, visualization, and modeling. We equipped Orange with components that use pre-trained deep convolutional networks to profile images with vectors of features. These vectors are used in image clustering and classification in a framework that enables mining of image sets for both novel and experienced users. We demonstrate the utility of the tool in image analysis of progenitor cells in mouse bone healing, identification of developmental competence in mouse oocytes, subcellular protein localization in yeast, and developmental morphology of social amoebae.

Democratized image analytics by visual programming through integration of deep models and small-scale machine learning.

Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7 SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1 SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1 SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7 SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.

Dongsu Park

Center for Regenerative Medicine,
Massachusetts General Hospital,
Center for Regenerative Medicine, Massachusetts General Hospital

Harvard Stem Cell Institute

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Dongsu Park

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Regenerative Medicine,Massachusetts General Hospital,Center for Regenerative Medicine, Massachusetts General Hospital

Harvard Stem Cell Institute

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Center for Regenerative Medicine,
Massachusetts General Hospital,
Center for Regenerative Medicine, Massachusetts General Hospital