Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

The Children's Hospital of Philadelphia

Poikiloderma &#x597D;&#x4E2D;&#x7403;&#x6E1B;&#x5C11;&#x75C7; (PN) &#x3068; C16orf57 &#x306E;&#x5909;&#x7570;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x308B;&#x307E;&#x308C;&#x306A;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x306E;&#x52A3;&#x6027; genodermatosis &#x3067;&#x3059;&#x3002;17 &#x65E5;&#x306B; 31 &#x306E; PN &#x60A3;&#x8005;&#x3067;&#x5909;&#x7570;&#x3092;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

Poikiloderma &#x597D;&#x4E2D;&#x7403;&#x6E1B;&#x5C11;&#x75C7;&#x60A3;&#x8005;&#x306B;&#x304A;&#x3051;&#x308B;&#x65B0;&#x898F; C16orf57 &#x5909;&#x7570;: bioinformatic &#x306E;&#x5206;&#x6790;&#x306E;&#x86CB;&#x767D;&#x8CEA;&#x3068;&#x3059;&#x3079;&#x3066;&#x306E;&#x5831;&#x544A;&#x3055;&#x308C;&#x305F;&#x5909;&#x7570;&#x306E;&#x4E88;&#x6E2C;&#x52B9;&#x679C;&#x3002;

Poikiloderma &#xD638; &#xC911;&#xAD6C; &#xAC10;&#xC18C; &#xC99D; (PN)&#xC73C;&#xB85C; &#xB4DC;&#xBB38; &#xC0C1; &#xC5FC;&#xC0C9;&#xCCB4; &#xC5F4;&#xC131; genodermatosis C16orf57 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC758;&#xD574; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. 17 &#xC9C0;&#xAE08;&#xAE4C;&#xC9C0; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; 31 PN &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;.

Poikiloderma &#xD638; &#xC911;&#xAD6C; &#xAC10;&#xC18C; &#xC99D;&#xC744; &#xAC00;&#xC9C4; &#xD658;&#xC790;&#xC5D0; &#xC788;&#xB294; &#xC18C;&#xC124; C16orf57 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;: &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBAA8;&#xB4E0; &#xBCF4;&#xACE0; &#xB41C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xC608;&#xCE21;&#xB41C; &#xD6A8;&#xACFC; bioinformatic &#xBD84;&#xC11D;.

Poiquilodermia con Neutropenia (PN) es un genodermatosis recesivo de un autosoma raro causado por mutaciones de C16orf57. Hasta la fecha 17 mutaciones se han identificado en 31 pacientes de la PN.

C16orf57 mutaciones en pacientes con Poiquilodermia con Neutropenia: an&aacute;lisis bioinform&aacute;tico de la prote&iacute;na y los efectos previstos de todas las mutaciones divulgadas.

Poikiloderma &#x4E0E;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x51CF;&#x5C11; (PN) &#x662F;&#x9057;&#x4F20;&#x6027; C16orf57 &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5F15;&#x8D77;&#x7684;&#x7F55;&#x89C1;&#x5E38;&#x67D3;&#x8272;&#x4F53;&#x9690;&#x6027;&#x76AE;&#x80A4;&#x75C5;&#x3002;&#x81F3;&#x4ECA;&#x6709; 17 &#x4E2A;&#x7A81;&#x53D8;&#x5DF2; 31 PN &#x60A3;&#x8005;&#x3002;

Poikiloderma &#x4E0E;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x51CF;&#x5C11;&#x60A3;&#x8005;&#x7684;&#x65B0;&#x578B; C16orf57 &#x7A81;&#x53D8;&#xFF1A; &#x86CB;&#x767D;&#x8D28;&#x548C;&#x9884;&#x6D4B;&#x7684;&#x5F71;&#x54CD;&#x7684;&#x6240;&#x6709;&#x62A5;&#x544A;&#x7A81;&#x53D8;&#x7684;&#x751F;&#x7269;&#x4FE1;&#x606F;&#x5B66;&#x5206;&#x6790;&#x3002;

Poikiloderma with Neutropenia (PN) is a rare autosomal recessive genodermatosis caused by C16orf57 mutations. To date 17 mutations have been identified in 31 PN patients.

Novel C16orf57 mutations in patients with Poikiloderma with Neutropenia: bioinformatic analysis of the protein and predicted effects of all reported mutations.

Po&iuml;kilodermie avec neutrop&eacute;nie (PN) est une g&eacute;nodermatose autosomique r&eacute;cessive rare, due &agrave; des mutations de C16orf57. &Agrave; ce jour 17 mutations ont &eacute;t&eacute; identifi&eacute;es chez 31 patients PN.

Roman C16orf57 mutations chez les patients avec po&iuml;kilodermie neutrop&eacute;niques : analyse bioinformatique des prot&eacute;ines et des effets pr&eacute;vus de toutes les mutations signal&eacute;es.

Poikilodermie mit Neutropenie (PN) ist eine seltene autosomal-rezessiv-Genodermatose C16orf57 Mutationen. Bisher 17 Mutationen in 31 PN-Patienten wurden.

Roman C16orf57-Mutationen bei Patienten mit Poikilodermie mit Neutropenie: bioinformatische Analyse der Protein und prognostizierten Auswirkungen des alle gemeldeten Mutationen.

Poichiloderma con Neutropenia (PN) &egrave; una genodermatosi recessiva autosomal rara causata da mutazioni C16orf57. Ad oggi 17 sono state identificate mutazioni in 31 pazienti PN.

Romanzo C16orf57 mutazioni nei pazienti affetti da poichiloderma con Neutropenia: analisi bioinformatica della proteina e del previsti gli effetti di tutte le mutazioni segnalati.

Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo-generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo-generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo-generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo-derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.

Challenges and promises for the development of donor-independent platelet transfusions.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children initiated by mutations that deregulate cytokine receptor signaling. Studies of JMML are constrained by limited access to patient tissues. We generated induced pluripotent stem cells (iPSCs) from malignant cells of two JMML patients with somatic heterozygous p.E76K missense mutations in PTPN11, which encodes SHP-2, a non-receptor tyrosine phosphatase. In vitro differentiation of JMML iPSCs produced myeloid cells with increased proliferative capacity, constitutive activation of granulocyte-macrophage colony stimulating factor (GM-CSF) and enhanced STAT5/ERK phosphorylation, similar to primary JMML cells from patients. Pharmacological inhibition of MEK kinase in iPSC-derived JMML cells reduced their GM-CSF independence, providing rationale for a potential targeted therapy. Our studies offer renewable sources of biologically relevant human cells in which to explore the pathophysiology and treatment of JMML. More generally, we illustrate the utility of iPSCs for in vitro modeling of a human malignancy.

Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia.

Leuc&eacute;mie my&eacute;lomonocytaire juv&eacute;nile (JMML) est un n&eacute;oplasme my&eacute;loprolif&eacute;ratif agressif de la petite enfance, initi&eacute; par des mutations qui d&eacute;r&eacute;glementer signalisation de r&eacute;cepteurs de cytokines. &Eacute;tudes de JMML sont limit&eacute;s par l&#39;acc&egrave;s limit&eacute; aux tissus du patient. Nous avons g&eacute;n&eacute;r&eacute; des cellules souches pluripotentes induites (CISP) de cellules malignes de deux patients JMML avec p.E76K h&eacute;t&eacute;rozygotes somatique des mutations faux-sens PTPN11, qui code une non r&eacute;cepteurs tyrosine phosphatase SHP-2. La diff&eacute;renciation in vitro de JMML CISP produit des cellules my&eacute;lo&iuml;des avec augmentation de la capacit&eacute; prolif&eacute;rative, activation constitutive de granulocyte-macrophage colony stimulating factor (GM-CSF) et am&eacute;lior&eacute; la phosphorylation de STAT5/ERK, semblable &agrave; des cellules primaires de JMML provenant de patients. Inhibition pharmacologique de la kinase MEK en cellules JMML iPSC r&eacute;duit leur ind&eacute;pendance de GM-CSF, pr&eacute;voyant une th&eacute;rapie cibl&eacute;e potentielle de raison d&#39;&ecirc;tre. Nos &eacute;tudes offrent des sources d&#39;&eacute;nergie renouvelables des cellules biologiquement pertinentes permettant d&#39;&eacute;tudier la physiopathologie et le traitement des JMML. Plus g&eacute;n&eacute;ralement, nous illustrons l&#39;utilit&eacute; du CISP pour la mod&eacute;lisation d&#39;une malignit&eacute; humaine in vitro.

Cellules souches pluripotentes induites patient d&eacute;riv&eacute; r&eacute;capituler les anomalies h&eacute;matopo&iuml;&eacute;tiques de leuc&eacute;mie my&eacute;lomonocytaire juv&eacute;nile.

Leucemia mielomonocitica giovanile (JMML) &egrave; una neoplasia mieloproliferativa aggressivo dei bambini avviata da mutazioni che deregolamentare la segnalazione del ricevitore di cytokine. Studi di JMML sono vincolati da un accesso limitato ai tessuti paziente. Abbiamo generato cellule staminali pluripotenti indotte (iPSCs) da cellule maligne di due pazienti JMML con mutazioni missenso somatiche p.E76K eterozigoti in PTPN11, che codifica per una fosfatasi non-recettore tirosina SHP-2. Differenziazione in vitro di JMML iPSCs prodotte cellule mieloidi con maggiore capacit&agrave; proliferativa, attivazione costitutiva della colonia di granulociti-macrofagi stimolante factor (GM-CSF) e migliorate la fosforilazione STAT5/ERK, simile a cellule primarie di JMML da pazienti. Inibizione farmacologica della chinasi MEK in cellule JMML iPSC derivate ridotto loro indipendenza GM-CSF, che fornisce la logica per una potenziale terapia mirata. I nostri studi di offrire fonti rinnovabili delle cellule umane biologicamente rilevanti in cui esplorare la patofisiologia e trattamento di JMML. Pi&ugrave; in generale, ci illustrano l&#39;utilit&agrave; delle iPSCs per la modellazione in vitro di una malignit&agrave; umana.

Cellule staminali pluripotenti indotte derivato da paziente ripassare ematopoietiche anomalie di leucemia mielomonocitica giovanile.

Induced pluripotent stem cells (iPSCs) hold great promise for modeling human hematopoietic diseases. However, intrinsic variability in the capacities of different iPSC lines for hematopoietic development complicates comparative studies and is currently unexplained. We created and analyzed 3 separate iPSC clones from fibroblasts of 3 different normal individuals using a standardized approach that included excision of integrated reprogramming genes by Cre-Lox mediated recombination. Gene expression profiling and hematopoietic differentiation assays showed that independent lines from the same individual were generally more similar to one another than those from different individuals. However, one iPSC line (WT2.1) exhibited a distinctly different gene expression, proliferation rate, and hematopoietic developmental potential relative to all other iPSC lines. This "outlier" clone also acquired extensive copy number variations (CNVs) during reprogramming, which may be responsible for its divergent properties. Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential.

Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbβ3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbβ3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbβ3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC) system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41+CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP). Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

Spencer K. Sullivan

Division of Hematology

The Children's Hospital of Philadelphia

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Spencer K. Sullivan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Hematology

The Children's Hospital of Philadelphia

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Division of Hematology