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1.
Arterioscler Thromb Vasc Biol ; 37(11): 2007-2013, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28982668

RESUMO

Platelets, derived from megakaryocytes, are anucleate cytoplasmic discs that circulate in the blood stream and play major roles in hemostasis, inflammation, and vascular biology. Platelet transfusions are used in a variety of medical settings to prevent life-threatening thrombocytopenia because of cancer therapy, other causes of acquired or inherited thrombocytopenia, and trauma. Currently, platelets used for transfusion purposes are donor derived. However, there is a drive to generate nondonor sources of platelets to help supplement donor-derived platelets. Efforts have been made by many laboratories to generate in vitro platelets and optimize their production and quality. In vitro-derived platelets have the potential to be a safer, more uniform product, and genetic manipulation could allow for better treatment of patients who become refractory to donor-derived units. This review focuses on potential clinical applications of in vitro-derived megakaryocytes and platelets, current methods to generate and expand megakaryocytes from pluripotent stem cell sources, and the use of these cells for disease modeling.


Assuntos
Plaquetas/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Megacariócitos/fisiologia , Trombopoese , Plaquetas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Doenças Hematológicas/sangue , Doenças Hematológicas/genética , Doenças Hematológicas/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Megacariócitos/metabolismo , Megacariócitos/transplante , Fenótipo , Transfusão de Plaquetas
2.
Blood ; 130(2): 192-204, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28455282

RESUMO

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.


Assuntos
Plaquetas/citologia , Células da Medula Óssea/imunologia , Fator V/genética , Células Progenitoras de Megacariócitos/citologia , Megacariócitos/citologia , Animais , Apoptose/efeitos dos fármacos , Arteríolas/efeitos dos fármacos , Arteríolas/imunologia , Arteríolas/lesões , Biomarcadores/sangue , Plaquetas/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular , Linhagem da Célula/imunologia , Endocitose , Fator V/imunologia , Fator V/farmacologia , Citometria de Fluxo , Expressão Gênica , Humanos , Imunofenotipagem , Lasers , Células Progenitoras de Megacariócitos/imunologia , Megacariócitos/imunologia , Camundongos , Camundongos SCID , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia
3.
Stem Cell Reports ; 8(3): 589-604, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28196690

RESUMO

Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into ß-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant ß-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and ß-like cell functionality in humans.


Assuntos
Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Endoderma/efeitos dos fármacos , Endoderma/embriologia , Fator de Transcrição GATA6/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Modelos Biológicos , Família Multigênica , Mutação , Pâncreas/embriologia , Fenótipo , Tretinoína/farmacologia
4.
Blood ; 127(10): 1227-33, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26787738

RESUMO

Platelets are anucleate cytoplasmic discs derived from megakaryocytes that circulate in the blood and have major roles in hemostasis, thrombosis, inflammation, and vascular biology. Platelet transfusions are required to prevent the potentially life-threatening complications of severe thrombocytopenia seen in a variety of medical settings including cancer therapy, trauma, and sepsis. Platelets used in the clinic are currently donor-derived which is associated with concerns over sufficient availability, quality, and complications due to immunologic and/or infectious issues. To overcome our dependence on donor-derived platelets for transfusion, efforts have been made to generate in vitro-based platelets. Work in this area has advanced our understanding of the complex processes that megakaryocytes must undergo to generate platelets both in vivo and in vitro. This knowledge has also defined the challenges that must be overcome to bring in vitro-based platelet manufacturing to a clinical reality. This review will focus on our understanding of committed megakaryocytes and platelet release in vivo and in vitro, and how this knowledge can guide the development of in vitro-derived platelets for clinical application.


Assuntos
Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Megacariócitos/metabolismo , Transfusão de Plaquetas , Animais , Doadores de Sangue , Plaquetas/citologia , Humanos , Megacariócitos/citologia
5.
Methods Mol Biol ; 1353: 13-23, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25630922

RESUMO

Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50 %, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.


Assuntos
Doxiciclina/farmacologia , Marcação de Genes/métodos , Vetores Genéticos/química , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Transgenes , Dedos de Zinco/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Endonucleases/genética , Endonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter , Loci Gênicos , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/efeitos dos fármacos
6.
Blood ; 125(23): 3627-36, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25852052

RESUMO

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application.


Assuntos
Plaquetas , Macrófagos , Megacariócitos , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Linhagem Celular , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/transplante , Camundongos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombopoese
7.
Stem Cell Res ; 12(2): 441-51, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24412757

RESUMO

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.


Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Megacariócitos/citologia , Camundongos
8.
Diabetes ; 60(6): 1813-22, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21617188

RESUMO

OBJECTIVE: Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K(+) channel (K(ATP) channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. A mutation at the same residue (E1506K) was previously shown to cause congenital hyperinsulinemia. We sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia. RESEARCH DESIGN AND METHODS: Neonatal diabetic patients were sequenced for mutations in ABCC8 (SUR1) and KCNJ11 (Kir6.2). Wild-type and mutant K(ATP) channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS: Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell K(ATP) currents than wild-type, consistent with the patients' diabetes. Conversely, no E1506K currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. K(ATP) channels are activated by Mg-nucleotides (via SUR1) and blocked by ATP (via Kir6.2). All mutations decreased channel activation by MgADP but had little effect on MgATP activation, as assessed using an ATP-insensitive Kir6.2 subunit. Importantly, using wild-type Kir6.2, a 30-s preconditioning exposure to physiological MgATP concentrations (>300 µmol/L) caused a marked reduction in the ATP sensitivity of neonatal diabetic channels, a small decrease in that of wild-type channels, and no change for E1506K channels. This difference in MgATP inhibition may explain the difference in resting whole-cell currents found for the neonatal diabetes and hyperinsulinemia mutations. CONCLUSIONS: Mutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. Differentially altered nucleotide regulation by NBD2 of SUR1 can explain the respective clinical phenotypes.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Diabetes Mellitus/genética , Ácido Glutâmico/genética , Hiperinsulinismo/genética , Canais KATP/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/genética , Animais , Diabetes Mellitus/etiologia , Eletrofisiologia , Humanos , Hiperinsulinismo/etiologia , Recém-Nascido , Masculino , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Ratos , Receptores de Sulfonilureias , Xenopus laevis
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