the-production-pluripotent-stem-cells-from-mouse-amniotic-fluid-cells

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Mount Sinai Hospital

Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies.

Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis.

Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr(+)/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr(c) heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1(b)) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr(+)/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr(+)/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.

"Agouti NOD": identification of a CBA-derived Idd locus on Chromosome 7 and its use for chimera production with NOD embryonic stem cells.

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

Virus-free induction of pluripotency and subsequent excision of reprogramming factors.

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.

The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source of human fibroblasts is essential for long-term xeno-free hESC maintenance.

Human embryonic fibroblasts support single cell enzymatic expansion of human embryonic stem cells in xeno-free cultures.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) allows the derivation of -personalized stem cells. Transposon transgenesis is a novel and viable alternative to viral transduction methods for the delivery of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) to somatic cells. Since transposons can be introduced as naked DNA using common plasmid transfection protocols, they provide a safer alternative to viral methods. piggyBac transposons are host-factor independent and integrate stably into the target genome, yet benefit from the unique characteristic of seamless removal mediated by transient expression of piggyBac transposase. Thus, piggyBac transposition provides an effective means to generate human, transgene-free iPSCs. The protocol describes the production of iPSCs from human embryonic fibroblasts, delivering reprogramming factors via plasmid transfection and piggyBac transposition.

Transgene-free production of pluripotent stem cells using piggyBac transposons.

&#x56FD;&#x9645;&#x5E72;&#x7EC6;&#x80DE;&#x4E3B;&#x52A8;&#x5206;&#x6790; 125 (ES) &#x7684;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7CFB;&#x548C; 11 &#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72; (iPS) &#x5E72;&#x7EC6;&#x80DE;&#x7CFB;&#xFF0C;&#x6765;&#x81EA;&#x4E16;&#x754C;&#x5404;&#x5730;&#x7684;&#x6587;&#x5316;&#x8FC7;&#x7A0B;&#x4E2D;&#x53D1;&#x751F;&#x7684;&#x57FA;&#x56E0;&#x53D8;&#x5316; 38 &#x5B9E;&#x9A8C;&#x5BA4;&#x3002;&#x591A;&#x6570;&#x7EBF;&#x8DEF;&#x5206;&#x6790;&#x5728;&#x65E9;&#x671F;&#x548C;&#x665A;&#x671F;&#x7684;&#x901A;&#x9053;&#x3002;&#x5355;&#x6838;&#x82F7;&#x9178;&#x591A;&#x6001;&#x6027; (SNP) &#x7684;&#x5206;&#x6790;&#x8868;&#x660E;&#x5B83;&#x4EEC;&#x5305;&#x62EC;&#x5927;&#x591A;&#x6570;&#x4E3B;&#x8981;&#x65CF;&#x88D4;&#x7FA4;&#x4F53;&#x7684;&#x4EE3;&#x8868;&#x3002;&#x591A;&#x6570;&#x7EBF;&#x8DEF;&#x4ECD;&#x7136;&#x4FDD;&#x6301; karyotypically &#x6B63;&#x5E38;&#xFF0C;&#x4F46;&#x6709;&#x8FDB;&#x6B65;&#x7684;&#x503E;&#x5411;&#xFF0C;&#x4EE5;&#x83B7;&#x5F97;&#x957F;&#x671F;&#x7684;&#x6587;&#x5316;&#xFF0C;&#x5E38;&#x53D7;&#x5F71;&#x54CD;&#x53F7; 1&#x3001; 12&#x3001; 17 &#x548C; 20 &#x53F7;&#x67D3;&#x8272;&#x4F53;&#x4E0A;&#x7684;&#x66F4;&#x6539;&#x3002;DNA &#x7532;&#x57FA;&#x5316;&#x6A21;&#x5F0F;&#x4E0E;&#x6CA1;&#x6709;&#x94FE;&#x63A5;&#x5230;&#x6587;&#x5316;&#x4E2D;&#x7684;&#x65F6;&#x95F4;&#x64C5;&#x81EA;&#x66F4;&#x6539;&#x3002;&#x7ED3;&#x6784;&#x7684;&#x53D8;&#x5F02;&#xFF0C;&#x4ECE;&#x5355;&#x6838;&#x82F7;&#x9178;&#x591A;&#x6001;&#x6027;&#x7684;&#x6570;&#x7EC4;&#xFF0C;&#x786E;&#x5B9A;&#x5076;&#x5C14;&#x4E5F;&#x51FA;&#x73B0;&#x4E86;&#x3002;&#x6CA1;&#x6709;&#x5171;&#x540C;&#x7684;&#x53D8;&#x5F02;&#x4E0E;&#x6587;&#x5316;&#x76F8;&#x5173;&#x7684;&#x88AB;&#x89C2;&#x5BDF;&#x5230;&#x67D3;&#x8272;&#x4F53; 1&#x3001; 12 &#x548C; 17&#xFF0C;&#x4F46;&#x6700;&#x5C11;&#x6269;&#x589E;&#x5B50;&#x5728;&#x67D3;&#x8272;&#x4F53; 20q11.21&#xFF0C;&#x5305;&#x62EC;&#x4E09;&#x4E2A;&#x57FA;&#x56E0;&#x5728;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#xFF0C;ID1&#x3001; BCL2L1 &#x548C; HM13&#xFF0C;&#x8868;&#x793A;&#x53D1;&#x751F;&#x5728; &gt; &#x7EBF;&#x7684; 20%&#x3002;&#x8FD9;&#x4E9B;&#x57FA;&#x56E0;&#x7684; BCL2L1 &#x9A7E;&#x9A76;&#x6587;&#x5316;&#x9002;&#x5E94;&#x7684;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x662F;&#x5F3A;&#x6709;&#x529B;&#x7684;&#x4EBA;&#x9009;&#x3002;

&#x4E0D;&#x540C;&#x79CD;&#x65CF;&#x7684;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x7B5B;&#x9009;&#x6807;&#x8BC6; 20 &#x53F7;&#x67D3;&#x8272;&#x4F53;&#x6700;&#x5C0F;&#x6269;&#x589E;&#x5B50;&#x8D4B;&#x4E88;&#x751F;&#x957F;&#x4F18;&#x52BF;&#x3002;

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

L&#x26;#39;Initiative internationale sur les cellules souches embryonnaires humaines analys&#xE9; 125 lignes de cellules souches (ES) et 11 induits pluripotentes lignes de cellules souches (iPS), &#xE0; partir de 38 laboratoires &#xE0; travers le monde, pour des modifications g&#xE9;n&#xE9;tiques qui se produisent pendant la culture. La plupart des lignes ont &#xE9;t&#xE9; analys&#xE9;s &#xE0; un passage pr&#xE9;coce et tardive. Un seul nucl&#xE9;otide polymorphisme (SNP) analyse a r&#xE9;v&#xE9;l&#xE9; qu&#x26;#39;ils comprenaient des repr&#xE9;sentants de la plupart des principaux groupes ethniques. La plupart des lignes est rest&#xE9;e caryotype normal, mais il y avait une tendance progressive &#xE0; acqu&#xE9;rir des changements sur la culture prolong&#xE9;e, qui influencent g&#xE9;n&#xE9;ralement les chromosomes 1, 12, 17 et 20. M&#xE9;thylation de l&#x26;#39;ADN modifi&#xE9; au hasard sans lien avec le temps dans la culture. Variantes structurelles, d&#xE9;termin&#xE9;es &#xE0; partir des tableaux SNP, est &#xE9;galement apparu de fa&#xE7;on sporadique. Aucun des variants communs li&#xE9;s &#xE0; la culture ont &#xE9;t&#xE9; observ&#xE9;s sur les chromosomes 1, 12 et 17, mais un amplicon minimale au niveau du chromosome 20q11.21, dont trois g&#xE8;nes exprim&#xE9;s dans les cellules souches embryonnaires humaines, ID1, BCL2L1 et HM13, se sont produites chez&#x26;gt; 20% des lignes. Parmi ces g&#xE8;nes, BCL2L1 est un candidat solide pour la conduite d&#x26;#39;adaptation de la culture de cellules souches embryonnaires.

D&#xE9;pistage et diversit&#xE9; ethnique cellules souches embryonnaires humaines Identifie un Chromosome 20 avantage minime croissance Amplicon Conf&#xE9;rer

International Stem Cell Initiative analysiert 125 menschlichen embryonalen Stammzellen (ES)-Zelllinien und 11 induzierten pluripotenten Stammzellen (iPS) Zelllinien aus 38 Labors weltweit, f&uuml;r genetische Ver&auml;nderungen w&auml;hrend der Kultur. Die meisten Linien wurden auf eine fr&uuml;he und sp&auml;te Passage analysiert. Single Nucleotide Polymorphism (SNP) Analyse ergab, dass sie Vertreter der meisten ethnische Gruppen eingebunden. Die meisten Linien blieb karyotypisch normal, aber es gab eine progressive Tendenz &Auml;nderungen auf l&auml;ngere Kultur, die der Chromosomen 1, 12, 17 und 20 Regel beeinflussen zu erwerben. DNA-Methylierungsmuster &auml;nderte sich planlos mit keine Verbindung zu Zeit in Kultur. Strukturelle Varianten, ermittelt aus der SNP-Arrays, erschien auch sporadisch. Keine gemeinsamen Variante mit Bezug zu Kultur auf Chromosomen 1, 12 und 17 beobachtet wurden, aber ereignete sich eine minimale Amplikons in Chromosom 20q11.21, einschlie&szlig;lich drei Genen ausgedr&uuml;ckt in menschlichen ES Zellen, ID1, BCL2L1 und HM13, &gt; 20 % der Leitungen. Dieser Gene ist BCL2L1 ein starker Kandidat f&uuml;r die Fahrt von Kultur-Adaption von ES-Zellen.

Screening ethnische Vielfalt menschlichen embryonalen Stammzellen identifiziert ein Chromosom 20 minimal Amplikons Gemeinschaftspatentsachen Wachstum nutzen.

L&#39;iniziativa internazionale sulle cellule staminali analizzato 125 linee di cellule staminali embrionali umane (ES) e 11 pluripotenti indotte staminali (iPS) linee cellulari, 38 laboratori in tutto il mondo, per mutazioni genetiche che si verificano durante la cultura. La maggior parte delle linee sono state analizzate in un passaggio precoce e tardo. Analisi del polimorfismo a singolo nucleotide (SNP) ha rivelato che hanno incluso i rappresentanti della maggior parte dei principali gruppi etnici. La maggior parte delle linee rimasero aberrazioni normale, ma c&#39;&egrave; stata una progressiva tendenza ad acquisire le modifiche su coltura prolungata, che colpiscono di solito cromosomi 1, 12, 17 e 20. Schemi di metilazione del DNA modificati a casaccio senza alcun collegamento a tempo in cultura. Varianti strutturali, determinati da matrici di SNP, apparvero sporadicamente. No varianti comuni legati alla cultura sono state osservate sui cromosomi 1, 12 e 17, ma un minimo amplicon nel cromosoma 20q11.21, tra cui tre geni espressi nelle cellule ES umane, ID1, BCL2L1 e HM13, si &egrave; verificato &amp;gt; 20% delle linee. Di questi geni, BCL2L1 &egrave; un forte candidato per la guida di adattamento della coltura delle cellule di ES.

Cellule staminali embrionali umane etnicamente diversificate di screening identifica un vantaggio di crescita cromosoma 20 conferimento minimo amplicon.

&#x56FD;&#x969B;&#x5E79;&#x7D30;&#x80DE;&#x30A4;&#x30CB;&#x30B7;&#x30A2;&#x30C1;&#x30D6;&#x306F;&#x3001;&#x57F9;&#x990A;&#x4E2D;&#x306B;&#x767A;&#x751F;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x7684;&#x5909;&#x5316;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E16;&#x754C;38&#x6A5F;&#x95A2;&#x304B;&#x3089;&#x3001;125&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#xFF08;ES&#xFF09;&#x7D30;&#x80DE;&#x682A;&#x3068;11&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#xFF08;iPS&#xFF09;&#x7D30;&#x80DE;&#x682A;&#x3092;&#x89E3;&#x6790;&#x3057;&#x305F;&#x3002;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x884C;&#x306F;&#x3001;&#x521D;&#x671F;&#x304A;&#x3088;&#x3073;&#x5F8C;&#x671F;&#x306E;&#x901A;&#x8DEF;&#x306B;&#x5206;&#x6790;&#x3057;&#x305F;&#x3002;&#x4E00;&#x5869;&#x57FA;&#x591A;&#x578B;&#xFF08;SNP&#xFF09;&#x89E3;&#x6790;&#x306F;&#x3001;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x4E3B;&#x8981;&#x306A;&#x6C11;&#x65CF;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x4EE3;&#x8868;&#x8005;&#x304C;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x884C;&#x306F;&#x3001;&#x6838;&#x578B;&#x6B63;&#x5E38;&#x3067;&#x3042;&#x3063;&#x305F;&#x304C;&#x3001;&#x4E00;&#x822C;&#x7684;&#x306B;&#x67D3;&#x8272;&#x4F53;1&#x3001;12&#x3001;17&#x3068;20&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x3001;&#x9577;&#x671F;&#x57F9;&#x990A;&#x3067;&#x5909;&#x66F4;&#x3092;&#x53D6;&#x5F97;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x9032;&#x6B69;&#x7684;&#x306A;&#x50BE;&#x5411;&#x304C;&#x3042;&#x3063;&#x305F;&#x3002; DNA&#x306E;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x3001;&#x57F9;&#x990A;&#x4E2D;&#x306E;&#x6642;&#x9593;&#x3078;&#x306E;&#x30EA;&#x30F3;&#x30AF;&#x306A;&#x3057;&#x3067;&#x7121;&#x8A08;&#x753B;&#x306B;&#x5909;&#x66F4;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002; SNP&#x30A2;&#x30EC;&#x30A4;&#x304B;&#x3089;&#x6C7A;&#x5B9A;&#x69CB;&#x9020;&#x5909;&#x7570;&#x4F53;&#x3082;&#x3001;&#x6563;&#x767A;&#x7684;&#x306B;&#x767B;&#x5834;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6587;&#x5316;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x4E00;&#x5207;&#x306E;&#x4E00;&#x822C;&#x7684;&#x306A;&#x5909;&#x7570;&#x4F53;&#x306F;&#x3001;&#x67D3;&#x8272;&#x4F53;1&#x3001;12&#x65E5;&#x3001;17&#x65E5;&#x306B;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x306A;&#x304B;&#x3063;&#x305F;&#x304C;&#x3001;3&#x3064;&#x306E;&#x30D2;&#x30C8;ES&#x7D30;&#x80DE;&#x3067;&#x767A;&#x73FE;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x3001;ID1&#x3001;BCL2L1&#x3068;HM13&#x3092;&#x542B;&#x3080;&#x67D3;&#x8272;&#x4F53;20q11.21&#x3001;&#x6700;&#x5C0F;&#x9650;&#x306E;&#x30A2;&#x30F3;&#x30D7;&#x30EA;&#x30B3;&#x30F3;&#x306F;&#x3001;&#x884C;&#x306E;&#x26;gt; 20&#xFF05;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002;&#x305F;&#x3053;&#x308C;&#x3089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x3001;BCL2L1&#x306F;&#x3001;ES&#x7D30;&#x80DE;&#x306E;&#x57F9;&#x990A;&#x9069;&#x5FDC;&#x3092;&#x99C6;&#x52D5;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x6709;&#x529B;&#x306A;&#x5019;&#x88DC;&#x3067;&#x3059;&#x3002;

&#x6C11;&#x65CF;&#x7684;&#x306B;&#x591A;&#x69D8;&#x306A;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3059;&#x308B;&#x3068;&#x3001;&#x6210;&#x9577;&#x306E;&#x512A;&#x4F4D;&#x6027;&#x3092;&#x4ED8;&#x4E0E;&#x3059;&#x308B;&#x7B2C;20&#x67D3;&#x8272;&#x4F53;&#x6700;&#x5C0F;&#x9650;&#x306E;&#x30A2;&#x30F3;&#x30D7;&#x30EA;&#x30B3;&#x30F3;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;

&#xAD6D;&#xC81C; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC0AC;&#xC5C5; &#xBD84;&#xC11D; 125 &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; (ES&#xC758;) &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xACFC; 38 &#xC2E4;&#xD5D8;&#xC2E4; &#xBB38;&#xD654; &#xC911;&#xC5D0; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC804;&#xC138;&#xACC4;&#xC5D0;&#xC11C; 11 &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; (iPS) &#xC138;&#xD3EC; &#xB77C;&#xC778;. &#xB300;&#xBD80;&#xBD84;&#xC758; &#xB77C;&#xC778;&#xC740; &#xC77C;&#xCC0D; &#xD558; &#xACE0; &#xB2A6;&#xAC8C; &#xD1B5;&#xB85C;&#xC5D0;&#xC11C; &#xBD84;&#xC11D; &#xD588;&#xB2E4;. &#xB2E8;&#xC77C; &#xC5FC;&#xAE30; &#xB2E4;&#xD615;&#xC131; (SNP) &#xBD84;&#xC11D;&#xC740; &#xB300;&#xBD80;&#xBD84;&#xC758; &#xC8FC;&#xC694; &#xC18C;&#xC218; &#xBBFC;&#xC871; &#xADF8;&#xB8F9;&#xC758; &#xB300;&#xD45C; &#xD3EC;&#xD568; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xB300;&#xBD80;&#xBD84;&#xC758; &#xB77C;&#xC778; karyotypically &#xC815;&#xC0C1; &#xB0A8;&#xC544; &#xC788;&#xC9C0;&#xB9CC; &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xACBD;&#xD5A5;&#xC774; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xC5FC;&#xC0C9;&#xCCB4; 1, 12, 17 &#xBC0F; 20&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC7A5;&#xAE30; &#xBB38;&#xD654;&#xC5D0; &#xBCC0;&#xD654;&#xB97C; &#xD68D;&#xB4DD; &#xD588;&#xB2E4;. DNA &#xBA54; &#xD2F8; &#xD654; &#xD328;&#xD134; &#xC544;&#xBB34; &#xB9C1;&#xD06C;&#xC640; &#xD568;&#xAED8; &#xBB38;&#xD654;&#xC5D0; &#xC2DC;&#xAC04; &#xB418;&#xB294; &#xB300;&#xB85C; &#xBCC0;&#xACBD; &#xD569;&#xB2C8;&#xB2E4;. SNP &#xBC30;&#xC5F4;&#xC5D0;&#xC11C; &#xACB0;&#xC815; &#xAD6C;&#xC870;&#xC801; &#xBCC0;&#xD615; &#xB610;&#xD55C; &#xC0B0;&#xBC1C;&#xC801;&#xC73C;&#xB85C; &#xB4F1;&#xC7A5;. &#xC544;&#xB2C8; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xBCC0;&#xD615; &#xBB38;&#xD654;&#xC5D0; &#xAD00;&#xB828; &#xB41C; 1, 12, 17, &#xC5FC;&#xC0C9;&#xCCB4;&#xC5D0; &#xAD00;&#xCC30; &#xB418;&#xC9C0; &#xC54A;&#xC558;&#xB2E4; &#xD558;&#xC9C0;&#xB9CC; &#xC5FC;&#xC0C9;&#xCCB4; 20q11.21, ID1, BCL2L1 &#xBC0F; HM13, &#xC778;&#xAC04;&#xC758; ES &#xC138;&#xD3EC;&#xC5D0; &#xD45C;&#xD604; &#xD558;&#xB294; 3 &#xAC1C;&#xC758; &#xC720;&#xC804;&#xC790;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC;&#xC5D0;&#xC11C; &#xCD5C;&#xC18C; amplicon&#xC5D0; &#xBC1C;&#xC0DD; &#xD55C; &amp;gt; &#xB77C;&#xC778;&#xC758; 20%. &#xC774; &#xC720;&#xC804;&#xC790;&#xC758; BCL2L1 ES &#xC138;&#xD3EC;&#xC758; &#xBB38;&#xD654; &#xC801;&#xC751; &#xC6B4;&#xC804;&#xC5D0; &#xB300; &#xD55C; &#xAC15;&#xD55C; &#xD6C4;&#xBCF4;&#xC785;&#xB2C8;&#xB2E4;.

&#xB2E4;&#xC591; &#xD55C; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC2EC;&#xC0AC; &#xC5FC;&#xC0C9;&#xCCB4; 20 &#xCD5C;&#xC18C; amplicon &#xBD80;&#xC5EC; &#xC131;&#xC7A5; &#xC6B0;&#xC704;&#xB97C; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

La Iniciativa Internacional de C&#xE9;lulas Madre analizaron 125 madre embrionarias humanas (ES) y 11 l&#xED;neas de c&#xE9;lulas madre pluripotentes inducidas (iPS) a las l&#xED;neas celulares, procedentes de 38 laboratorios en todo el mundo, por los cambios gen&#xE9;ticos que ocurren durante el cultivo. La mayor&#xED;a de l&#xED;neas se analizaron en un pasaje temprana y tard&#xED;a. Polimorfismo de nucle&#xF3;tido &#xFA;nico (SNP), el an&#xE1;lisis revel&#xF3; que incluy&#xF3; a representantes de la mayor&#xED;a de los principales grupos &#xE9;tnicos. La mayor&#xED;a de las l&#xED;neas se mantuvo cariotipo normal, pero hubo una tendencia progresiva a la adquisici&#xF3;n de los cambios en la cultura prolongado, que suele afectar a los cromosomas 1, 12, 17 y 20. Los patrones de metilaci&#xF3;n del ADN cambia al azar sin ning&#xFA;n v&#xED;nculo con el tiempo en la cultura. Variantes estructurales, determinados a partir de las matrices SNP, tambi&#xE9;n aparecieron espor&#xE1;dicamente. No hay variantes comunes relacionadas con la cultura se observaron en los cromosomas 1, 12 y 17, pero un m&#xED;nimo de amplificaci&#xF3;n en el cromosoma 20q11.21, entre ellos tres genes que se expresan en c&#xE9;lulas madre embrionarias humanas, ID1, BCL2L1 y HM13, se produjeron en&#x26;gt; 20% de las l&#xED;neas. De estos genes, BCL2L1 es un fuerte candidato para la conducci&#xF3;n de la adaptaci&#xF3;n de cultivo de c&#xE9;lulas madre embrionarias.

La detecci&#xF3;n de diferentes etnias c&#xE9;lulas madre embrionarias humanas Identifica un cromosoma 20 Amplicon m&#xED;nimo Conferir ventaja de crecimiento

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.

Article	Year
Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis. Biotechnology and bioengineering\| PubMed ID: 14574685	2003
"Agouti NOD": identification of a CBA-derived Idd locus on Chromosome 7 and its use for chimera production with NOD embryonic stem cells. Mammalian genome : official journal of the International Mammalian Genome Society\| PubMed ID: 16261419	2005
Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature\| PubMed ID: 19252477	2009
piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature\| PubMed ID: 19252478	2009
Human embryonic fibroblasts support single cell enzymatic expansion of human embryonic stem cells in xeno-free cultures. Stem cell research\| PubMed ID: 20934930	2011
Transgene-free production of pluripotent stem cells using piggyBac transposons. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21822869	2011
Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nature biotechnology\| PubMed ID: 22119741	2011
Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis. Cell\| PubMed ID: 26875865	2016

Maria Mileikovsky

Biography

JoVE Journal Publications

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