zinc-finger-nuclease-enhanced-gene-targeting-human-embryonic-stem

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Monash University

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.

Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice.

The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag-pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of beta-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in beta-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdmicroDyIN), we were able to partially correct the mdx dystrophic phenotype. AdmicroDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%+/-6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdmicroDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.

Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle.

Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self-renewal and differentiation.

RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation.

&#x9AA8;&#x9AD3;&#x589E;&#x751F;&#x5F02;&#x5E38;&#x7EFC;&#x5408;&#x5F81; (MPS) &#x662F;&#x5F02;&#x6784;&#x7684; nonlymphoid &#x9020;&#x8840;&#x80BF;&#x7624;&#xFF0C;&#x88AB;&#x8BA4;&#x4E3A;&#x662F;&#x56FA;&#x6709;&#x7684;&#x9020;&#x8840;&#x7EC6;&#x80DE;&#xFF0C;&#x5B50;&#x7C7B;&#x3002;&#x5F3A;&#x5236;&#x6027;&#x516C;&#x79EF;&#x91D1;&#x8BA1;&#x5212;&#x7684;&#x539F;&#x56E0;&#x662F;&#x4E3B;&#x8981;&#x662F;&#x672A;&#x77E5;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E;&#x5C0F;&#x9F20;&#x89C6;&#x9EC4;&#x9178;&#x53D7;&#x4F53; &gamma; &#xFF08;RARgamma&#xFF09;&#xFF0C;&#x4E0D;&#x8DB3;&#x4EE5;&#x5E94;&#x4ED8;&#x53D1;&#x5C55;&#x5B8C;&#x5168;&#x7531; RARgamma &#x7F3A;&#x5FAE;&#x73AF;&#x5883;&#x8BF1;&#x5BFC;&#x7684;&#x56FD;&#x4F1A;&#x8BAE;&#x5458;&#x3002;RARgamma(-/-) &#x5C0F;&#x9F20;&#x5927;&#x5927;&#x589E;&#x52A0;&#x4E86;&#x7C92;&#x7EC6;&#x80DE;/&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x548C;&#x7C92;&#x7EC6;&#x80DE;&#x5728;&#x9AA8;&#x9AD3; (BM)&#x3001; &#x672B;&#x68A2;&#x8840;&#x548C;&#x813E;&#x3002;&#x5F3A;&#x5236;&#x6027;&#x516C;&#x79EF;&#x91D1;&#x8BA1;&#x5212;&#x8868;&#x578B;&#x6301;&#x7EED;&#x4E86;&#x8001;&#x9F20;&#x7684;&#x5BFF;&#x547D;&#x548C;&#x8001;&#x5E74;&#x5C0F;&#x9F20;&#x4E2D;&#x66F4;&#x4E3A;&#x660E;&#x663E;&#x3002;&#x610F;&#x5916;&#x7684;&#x662F;&#xFF0C;&#x79FB;&#x690D;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#x8FD9;&#x79CD;&#x75BE;&#x75C5;&#x5E76;&#x4E0D;&#x662F;&#x56FA;&#x6709;&#x7684;&#x9020;&#x8840;&#x7EC6;&#x80DE;&#x3002;BM &#x4ECE;&#x91CE;&#x751F;&#x578B;&#x5C0F;&#x9F20;&#x79FB;&#x690D;&#x5230;&#x5C0F;&#x9F20;&#x4E0E; RARgamma(-/-) &#x5FAE;&#x73AF;&#x5883;&#x8FC5;&#x901F;&#x5236;&#x5B9A; MPS&#xFF0C;&#x5F15;&#x81F4;&#x90E8;&#x5206;&#x5927;&#x5927;&#x63D0;&#x5347; TNFalpha RARgamma(-/-) &#x5C0F;&#x9F20;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x5728; nonhematopoietic &#x4E2D;&#x663E;&#x793A;&#x8FD9;&#x4E00;&#x635F;&#x5931;&#x7684; RARgamma &#x7ED3;&#x679C;&#x5355;&#x5143;&#x683C;&#x5185;&#x5728; MPS&#xFF0C;&#x63ED;&#x793A;&#x9020;&#x8840;&#x969C;&#x788D;&#x7684;&#x552F;&#x4E00;&#x539F;&#x56E0;&#x5FAE;&#x73AF;&#x5883;&#x7684;&#x80FD;&#x529B;&#x3002;

&#x89C6;&#x9EC4;&#x9178;&#x53D7;&#x4F53; &gamma; &#x7F3A;&#x9677;&#x81F4;&#x5FAE;&#x73AF;&#x5883;&#x8BF1;&#x5BFC;&#x7684;&#x9AA8;&#x9AD3;&#x589E;&#x751F;&#x5F02;&#x5E38;&#x7EFC;&#x5408;&#x75C7;&#x3002;

Myeloproliferative syndromes (MPS) are a heterogeneous subclass of nonlymphoid hematopoietic neoplasms which are considered to be intrinsic to hematopoietic cells. The causes of MPS are largely unknown. Here, we demonstrate that mice deficient for retinoic acid receptor gamma (RARgamma), develop MPS induced solely by the RARgamma-deficient microenvironment. RARgamma(-/-) mice had significantly increased granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, and spleen. The MPS phenotype continued for the lifespan of the mice and was more pronounced in older mice. Unexpectedly, transplant studies revealed this disease was not intrinsic to the hematopoietic cells. BM from wild-type mice transplanted into mice with an RARgamma(-/-) microenvironment rapidly developed the MPS, which was partially caused by significantly elevated TNFalpha in RARgamma(-/-) mice. These data show that loss of RARgamma results in a nonhematopoietic cell-intrinsic MPS, revealing the capability of the microenvironment to be the sole cause of hematopoietic disorders.

A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency.

Syndromes my&#xE9;loprolif&#xE9;ratifs (SMP) sont une sous-classe h&#xE9;t&#xE9;rog&#xE8;ne de non lympho&#xEF;des n&#xE9;oplasmes h&#xE9;matopo&#xEF;&#xE9;tiques qui sont consid&#xE9;r&#xE9;s comme intrins&#xE8;que aux cellules h&#xE9;matopo&#xEF;&#xE9;tiques. Les causes de MPS sont largement inconnues. Ici, nous d&#xE9;montrons que les souris d&#xE9;ficientes pour l&#x26;#39;acide gamma-r&#xE9;cepteur r&#xE9;tino&#xEF;que (RARgamma), d&#xE9;velopper des MPS induites uniquement par le microenvironnement RARgamma-d&#xE9;ficient. RARgamma (- / -) des souris avait augment&#xE9; de fa&#xE7;on significative de granulocytes / macrophages et des granulocytes prog&#xE9;niteurs dans la moelle osseuse (BM), le sang p&#xE9;riph&#xE9;rique et la rate. Le ph&#xE9;notype MPS a continu&#xE9; pour la dur&#xE9;e de vie des souris et a &#xE9;t&#xE9; plus prononc&#xE9;e dans les souris plus &#xE2;g&#xE9;es. De fa&#xE7;on inattendue, des &#xE9;tudes de transplantation a r&#xE9;v&#xE9;l&#xE9; cette maladie n&#x26;#39;&#xE9;tait pas intrins&#xE8;que aux cellules h&#xE9;matopo&#xEF;&#xE9;tiques. BM &#xE0; partir de souris de type sauvage transplant&#xE9;s dans des souris avec un RARgamma (- / -) microenvironnement rapidement d&#xE9;velopp&#xE9; les MPS, qui a &#xE9;t&#xE9; partiellement caus&#xE9;es par le TNFalpha significativement &#xE9;lev&#xE9;e dans RARgamma (- / -) chez la souris. Ces donn&#xE9;es montrent que la perte des r&#xE9;sultats RARgamma dans un non h&#xE9;matopo&#xEF;&#xE9;tiques cellules intrins&#xE8;ques MPS, r&#xE9;v&#xE9;lant la capacit&#xE9; du micro-environnement pour &#xEA;tre la seule cause de troubles h&#xE9;matopo&#xEF;&#xE9;tiques.

Un syndrome my&#xE9;loprolif&#xE9;ratif induit par micro-environnement caus&#xE9;s par une carence en acide r&#xE9;tino&#xEF;que Receptor Gamma

Myeloproliferative Syndrome (MPS) sind eine heterogene Unterklasse von nonlymphoid h&auml;matopoetischen Tumoren, die betrachtet werden, blutbildender Zellen ansieht. Die Ursachen der MPS sind weitgehend unbekannt. Hier zeigen wir, dass M&auml;use f&uuml;r Retinoic S&auml;ure des Gammas (RARgamma), mangelhaft entwickeln MPS induzierte allein durch die RARgamma-defizienten Mikromilieu. RARgamma(-/-) M&auml;use hatte merklich Vorl&auml;uferzellen der Granulozyten/Makrophagen und Granulozyten im Knochenmark (BM), peripheres Blut und Milz. Der MPS-Ph&auml;notyp setzte f&uuml;r die Lebensdauer der M&auml;use und wurde bei &auml;lteren M&auml;usen st&auml;rker ausgepr&auml;gt. Unerwartet offenbart Transplantation Studien, dass diese Krankheit nicht immanent blutbildender Zellen war. BM von Wildtyp M&auml;usen transplantierte in M&auml;use mit einer RARgamma(-/-)-Mikromilieu rasch entwickelt MPS, die teilweise durch deutlich erh&ouml;hte TNFalpha bei RARgamma(-/-) M&auml;usen verursacht wurde. Diese Daten zeigen diesen Verlust der RARgamma Ergebnisse in eine Nonhematopoietic Zelle systeminterne MPS, offenbart die F&auml;higkeit das Mikromilieu zu sein, die Sohle h&auml;matopoetischer Erkrankungen verursachen.

Ein Mikromilieu induziert Myelodysplastische Syndrom durch Retinoic S&auml;ure-Rezeptor Gamma Mangel verursacht.

Sindromi mieloproliferative (MPS) sono una sottoclasse eterogenea di neoplasie emopoietiche nonlymphoid che sono considerati essere intrinseca alle cellule ematopoietiche. Le cause di MPS sono in gran parte sconosciute. Qui, ci dimostrano che topi carenti per gamma di recettore dell&#39;acido retinoico (RARgamma), sviluppare MPS indotte unicamente dal microambiente RARgamma-carenti. RARgamma(-/-) topi avevano aumentato significativamente progenitori granulociti/macrofagi e granulociti nel sangue periferico, midollo osseo (BM) e la milza. Il fenotipo MPS ha continuato per la durata di vita dei topi ed &egrave; stato pi&ugrave; pronunciato nei topi anziani. Inaspettatamente, gli studi di trapianto ha rivelato che questa malattia non era intrinseca alle cellule ematopoietiche. BM da selvaggio-tipo topi trapiantati in topi con un microambiente RARgamma(-/-) rapidamente sviluppato il MPS, che parzialmente &egrave; stata causata da TNFalpha significativamente elevato nei topi RARgamma(-/-). Questi dati mostrano che la perdita di RARgamma risultati in un non emopoietico MPS intrinseca delle cellule, rivelando la capacit&agrave; del microambiente di essere l&#39;unica causa di disordini ematopoietici.

Una microambiente indotta mieloproliferativa sindrome causata da deficit di gamma del recettore dell&#39;acido retinoico.

&#x9AA8;&#x9AC4;&#x5897;&#x6B96;&#x6027;&#x75C7;&#x5019;&#x7FA4;&#xFF08;MPS&#xFF09;&#x306F;&#x3001;&#x9020;&#x8840;&#x7D30;&#x80DE;&#x306B;&#x56FA;&#x6709;&#x3067;&#x3042;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x975E;&#x30EA;&#x30F3;&#x30D1;&#x7CFB;&#x9020;&#x8840;&#x5668;&#x816B;&#x760D;&#x306E;&#x7570;&#x7A2E;&#x306E;&#x30B5;&#x30D6;&#x30AF;&#x30E9;&#x30B9;&#x3067;&#x3059;&#x3002; MPS&#x306E;&#x539F;&#x56E0;&#x306F;&#x4E0D;&#x660E;&#x306A;&#x70B9;&#x304C;&#x591A;&#x3044;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30EC;&#x30C1;&#x30CE;&#x30A4;&#x30F3;&#x9178;&#x53D7;&#x5BB9;&#x4F53;&#x30AC;&#x30F3;&#x30DE;&#xFF08;RARgamma&#xFF09;&#x306E;&#x6B20;&#x640D;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;&#x3082;&#x3063;&#x3071;&#x3089;RARgamma&#x6B20;&#x640D;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306B;&#x3088;&#x308A;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x308B;MPS&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; RARgamma&#xFF08; -  /  - &#xFF09;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x6709;&#x610F;&#x306B;&#x9AA8;&#x9AC4;&#xFF08;BM&#xFF09;&#x3001;&#x672B;&#x68A2;&#x8840;&#x3001;&#x813E;&#x81D3;&#x306B;&#x9846;&#x7C92;&#x7403;/&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x304A;&#x3088;&#x3073;&#x9846;&#x7C92;&#x7403;&#x3092;&#x5897;&#x52A0;&#x3057;&#x305F;&#x3002; MPS&#x306E;&#x8868;&#x73FE;&#x578B;&#x306F;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5BFF;&#x547D;&#x3092;&#x7D99;&#x7D9A;&#x3057;&#x3001;&#x9AD8;&#x9F62;&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x3088;&#x308A;&#x9855;&#x8457;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002;&#x4E88;&#x671F;&#x305B;&#x305A;&#x3001;&#x79FB;&#x690D;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x75C5;&#x6C17;&#x306F;&#x9020;&#x8840;&#x7D30;&#x80DE;&#x306B;&#x56FA;&#x6709;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002; &#xFF08; -  /  - &#xFF09;RARgamma&#x3092;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x79FB;&#x690D;&#x3057;&#x305F;&#x91CE;&#x751F;&#x578B;&#x30DE;&#x30A6;&#x30B9;&#x306E;BM&#xFF08; -  /  - &#xFF09;&#x30DE;&#x30A6;&#x30B9;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306F;&#x6025;&#x901F;&#x306B;&#x90E8;&#x5206;&#x7684;&#x306B;RARgamma&#x3067;&#x6709;&#x610F;&#x306B;&#x4E0A;&#x6607;&#x3057;&#x305F;TNF&#x3B1;&#x306E;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x305F;MPS&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x975E;&#x9020;&#x8840;&#x7CFB;&#x7D30;&#x80DE;&#x56FA;&#x6709;&#x306E;MPS&#x3067;RARgamma&#x7D50;&#x679C;&#x306E;&#x640D;&#x5931;&#x306F;&#x3001;&#x9020;&#x8840;&#x6027;&#x75BE;&#x60A3;&#x306E;&#x552F;&#x4E00;&#x306E;&#x539F;&#x56E0;&#x3067;&#x3042;&#x308B;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

&#x30EC;&#x30C1;&#x30CE;&#x30A4;&#x30F3;&#x9178;&#x53D7;&#x5BB9;&#x4F53;&#x30AC;&#x30F3;&#x30DE;&#x6B20;&#x4E4F;&#x306B;&#x3088;&#x200B;&#x200B;&#x308B;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x306B;&#x3088;&#x3063;&#x3066;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x308B;&#x9AA8;&#x9AC4;&#x5897;&#x6B96;&#x6027;&#x75C7;&#x5019;&#x7FA4;

Myeloproliferative &#xC99D;&#xD6C4;&#xAD70; (MPS)&#xB294; &#xC870; &#xD608; &#xC138;&#xD3EC;&#xB97C; &#xBCF8;&#xC9C8;&#xC801;&#xC778; &#xAC83;&#xC73C;&#xB85C; &#xAC04;&#xC8FC; &#xB418;&#xB294; nonlymphoid &#xC870; &#xD608; neoplasms&#xC758; &#xB2E4;&#xB978; &#xC720;&#xD615;&#xC758; &#xD558;&#xC704; &#xD074;&#xB798;&#xC2A4;. MPS&#xC758; &#xC6D0;&#xC778;&#xC740; &#xD06C;&#xAC8C; &#xC54C;&#xB824;&#xC9C4;&#xC785;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, retinoic &#xC0B0; &#xC131; &#xC218;&#xC6A9; &#xCCB4; &#xAC10;&#xB9C8; (RARgamma)&#xC5D0; &#xB300; &#xD55C; &#xACB0;&#xD54D; &#xC0DD;&#xC950; &#xAC1C;&#xBC1C; MPS &#xC804;&#xC801;&#xC73C;&#xB85C; &#xBD88;&#xCDA9;&#xBD84; &#xD55C; RARgamma microenvironment&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB41C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. RARgamma(-/-) &#xC950; granulocyte/macrophage progenitors &#xBC0F; granulocytes (BM) &#xACE8; &#xC218;, &#xB9D0; &#xCD08; &#xD608;&#xC561; &#xBC0F; &#xBE44;&#xC7A5;&#xC5D0; &#xD06C;&#xAC8C; &#xC99D;&#xAC00; &#xD588;&#xB2E4;. MPS &#xD615; &#xC950;&#xC758; &#xC218;&#xBA85;&#xC5D0; &#xB300; &#xD55C; &#xACC4;&#xC18D; &#xD558; &#xACE0; &#xB354; &#xC624;&#xB798; &#xB41C; &#xC950;&#xC5D0;&#xC11C; &#xBC1C;&#xC74C; &#xB418;&#xC5C8;&#xB2E4;. &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xAC8C;, &#xC774;&#xC2DD; &#xC5F0;&#xAD6C; &#xBC1D;&#xD600;&#xC774; &#xBCD1; &#xC870; &#xD608; &#xC138;&#xD3EC;&#xB97C; &#xB0B4;&#xC7A5; &#xD588;&#xB2E4;. &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xB9C8;&#xC6B0;&#xC2A4; RARgamma(-/-) microenvironment&#xC640; &#xC950;&#xC5D0; &#xBE60;&#xB974;&#xAC8C; &#xC774;&#xC2DD;&#xC5D0;&#xC11C; BM &#xAC1C;&#xBC1C; RARgamma(-/-) &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0; &#xC0C1;&#xB2F9;&#xD788; &#xB192;&#xC740; Tnfalpha&#xC5D0; &#xAE30;&#xC778;&#xD55C; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xC758;&#xC6D0;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130;&#xB294; nonhematopoietic&#xC5D0;&#xC11C; RARgamma &#xACB0;&#xACFC;&#xC758; &#xC190;&#xC2E4; &#xD45C;&#xC2DC; &#xC140; &#xB0B4;&#xC7A5; MPS, &#xACC4;&#xC2DC;&#xB294; microenvironment &#xAE30;&#xB2A5;&#xC774; &#xB2E8;&#xB3C5; &#xC870; &#xD608; &#xC7A5;&#xC560;&#xC758; &#xC6D0;&#xC778;&#xC774; &#xB420;.

Microenvironment &#xC774;&#xC6A9;&#xD55C; myeloproliferative &#xC99D;&#xD6C4;&#xAD70; retinoic acid &#xC218;&#xC6A9; &#xCCB4; &#xAC10;&#xB9C8; &#xACB0;&#xD54D;&#xC5D0; &#xC758;&#xD574; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;.

S&#xED;ndromes mieloproliferativos (MPS) son una subclase heterog&#xE9;neo de neoplasias hematopoy&#xE9;ticas no linfoides que se consideran ser intr&#xED;nseco a las c&#xE9;lulas hematopoy&#xE9;ticas. Las causas de la MPS son en gran parte desconocido. Este sentido, demuestran que los ratones deficientes para gamma receptor de &#xE1;cido retinoico (RARgamma), desarrollar MPS inducidos &#xFA;nicamente por el microambiente RARgamma deficiente. RARgamma (- / -) ratones hab&#xED;an aumentado de manera significativa de granulocitos / macr&#xF3;fagos y granulocitos progenitores de m&#xE9;dula &#xF3;sea (MO), sangre perif&#xE9;rica y el bazo. El fenotipo MPS continu&#xF3; durante la vida &#xFA;til de los ratones y fue m&#xE1;s pronunciada en los ratones m&#xE1;s viejos. Inesperadamente, los estudios de trasplante revelado esta enfermedad no era intr&#xED;nseco a las c&#xE9;lulas hematopoy&#xE9;ticas. BM de ratones de tipo salvaje en ratones trasplantados con una RARgamma (- / -) microambiente desarrollado r&#xE1;pidamente el MPS, que fue causado en parte por TNF significativamente elevados en RARgamma (- / -) ratones. Estos datos muestran que la p&#xE9;rdida de los resultados RARgamma en una c&#xE9;lula no hematopoy&#xE9;ticas-intr&#xED;nsecas MPS, revelando la capacidad del microambiente a ser la &#xFA;nica causa de los trastornos hematopoy&#xE9;ticos.

Un s&#xED;ndrome mieloproliferativo Microambiente-, causadas por la deficiencia Gamma receptor del &#xE1;cido retinoico

LIM homeobox transcription factor 1 alpha (Lmx1a) is required for the development of midbrain dopaminergic neurons, roof plate formation, and cortical hem development. We generated a reporter embryonic stem cell (ESC) line for Lmx1a and used it to track differentiation and extract neural progenitors from differentiating mouse ESCs. Lmx1a(+) cells gave rise to functional cortical upper layer GABAergic neurons or dopaminergic neurons depending on the culture conditions used for differentiation. Under chemically defined neurobasal conditions, ESC differentiation resulted in widespread and transient expression of Lmx1a, without the addition of exogenous factors such as sonic hedgehog (Shh), Wnts, and/or bone morphogenic proteins (BMPs). Under neutral conditions, Lmx1a(+) cells express genes known to be downstream of Lmx1a and cortical hem markers Wnt3a and p73. The majority of these cells did not express the ventral midbrain dopaminergic marker Foxa2 or dorsal roof plate marker BMP-2. Lmx1a(+) -Foxa2(-) cells were primed to become SatB2(+) GABAergic neurons and appeared to be resistant to dopaminergic patterning cues. PA6 coculture produced a substantial population of Lmx1a(+) progenitors that also expressed Foxa2 and on further differentiation gave rise to dopaminergic neurons at high frequency. We conclude that Lmx1a is a useful marker for the extraction of progenitors of GABAergic or dopaminergic neurons. We caution against the assumption that it indicates dopaminergic commitment during in vitro differentiation of ESCs. Indeed, in monolayer culture under neurobasal conditions, with or without the addition of Shh and fibroblast growth factor 8 (FGF8), Lmx1a(+) cells were predominantly progenitors of forebrain GABAergic neurons. We obtained dopaminergic cells in large numbers only by coculture with PA6 cells.

Lmx1a allows context-specific isolation of progenitors of GABAergic or dopaminergic neurons during neural differentiation of embryonic stem cells.

The identification of small molecules capable of directing pluripotent cell differentiation towards specific lineages is highly desirable to both reduce cost, and increase efficiency. Within neural progenitors, LIM homeobox transcription factor 1 alpha (Lmx1a) is required for proper development of roof plate and cortical hem structures of the forebrain, as well as the development of floor plate and midbrain dopaminergic neurons. In this study we generated homologous recombinant cell lines expressing either luciferase or β-lactamase under the control of the Lmx1a promoter, and used these cell lines to investigate kinase-mediated regulation of Lmx1a activity during neuronal differentiation. A screen of 143 small molecule tyrosine kinase inhibitors yielded 16 compounds that positively or negatively modulated Lmx1a activity. Inhibition of EGF, VEGF and DNA-dependent protein kinase (DNA-PK) signaling significantly upregulated Lmx1a activity whereas MEK inhibition strongly downregulated its activity. Quantitative FACS analysis revealed that the DNA-PK inhibitor significantly increased the number of Lmx1a+ progenitors while subsequent qPCR showed an upregulation of Notch effectors, the basic helix-loop-helix genes, Hes5 and Hey1. FACS further revealed that DNA-PK-mediated regulation of Lmx1a+ cells is dependent on the rapamycin-sensitive complex, mTORC1. Interestingly, this DNA-PK inhibitor effect was preserved in a co-culture differentiation protocol. Terminal differentiation assays showed that DNA-PK inhibition shifted development of neurons from forebrain toward midbrain character as assessed by Pitx3/TH immunolabeling and corresponding upregulation of midbrain (En1), but not forebrain (FoxG1) transcripts. These studies show that Lmx1a signaling in mouse embryonic stem cells contributes to a molecular cascade establishing neuronal specification. The data presented here identifies a novel regulatory pathway where signaling from DNA-PK appears to suppress midbrain-specific Lmx1a expression.

Article	Year
Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre dege... Human molecular genetics\| PubMed ID: 11929846	2002
Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle. Human molecular genetics\| PubMed ID: 12095914	2002
RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation. The Journal of experimental medicine\| PubMed ID: 16682494	2006
A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell\| PubMed ID: 17574023	2007
Lmx1a allows context-specific isolation of progenitors of GABAergic or dopaminergic neurons during neural differentiation of embryonic stem cells. Stem cells (Dayton, Ohio)\| PubMed ID: 22495882	2012
DNA-dependent protein kinase is a context dependent regulator of Lmx1a and midbrain specification. PloS one\| PubMed ID: 24194952	2013

Stewart A. Fabb

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