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1.
Cell ; 177(4): 910-924.e22, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982595

RESUMO

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Análise de Célula Única , Via de Sinalização Wnt
2.
Cell ; 149(6): 1284-97, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22632761

RESUMO

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.


Assuntos
Antineoplásicos/farmacologia , Antagonistas de Dopamina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células-Tronco Neoplásicas/efeitos dos fármacos , Tioridazina/farmacologia , Animais , Citarabina/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Mefloquina/farmacologia , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Piranos/farmacologia
3.
Nature ; 560(7719): E32, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30042505

RESUMO

In this Article, there were duplicated empty lanes in Supplementary Figs. 2e and 3b. The corrected figures are presented in the Supplementary Information to the accompanying Amendment. The original Article has not been corrected.

4.
Stem Cells ; 35(9): 2095-2102, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28758276

RESUMO

Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102.


Assuntos
Reprogramação Celular , Hematopoese , Células-Tronco Pluripotentes Induzidas/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Células da Medula Óssea/patologia , Humanos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo
5.
Stem Cells ; 33(4): 1142-52, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25639500

RESUMO

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here, we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible, as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore, the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM, thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential, which can be modulated by culture conditions in a predictive and quantitative manner.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Animais , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
6.
Stem Cells ; 33(6): 1839-49, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25764124

RESUMO

Current treatments that use hematopoietic progenitor cell (HPC) transplantation in acute myeloid leukemia (AML) patients substantially reduce the risk of relapse, but are limited by the availability of immune compatible healthy HPCs. Although cellular reprogramming has the potential to provide a novel autologous source of HPCs for transplantation, the applicability of this technology toward the derivation of healthy autologous hematopoietic cells devoid of patient-specific leukemic aberrations from AML patients must first be evaluated. Here, we report the generation of human AML patient-specific hematopoietic progenitors that are capable of normal in vitro differentiation to myeloid lineages and are devoid of leukemia-associated aberration found in matched patient bone marrow. Skin fibroblasts were obtained from AML patients whose leukemic cells possessed a distinct, leukemia-associated aberration, and used to create AML patient-specific induced pluripotent stem cells (iPSCs). Through hematopoietic differentiation of AML patient iPSCs, coupled with cytogenetic interrogation, we reveal that AML patient-specific HPCs possess normal progenitor capacity and are devoid of leukemia-associated mutations. Importantly, in rare patient skin samples that give rise to mosaic fibroblast cultures that continue to carry leukemia-associated mutations; healthy hematopoietic progenitors can also be generated via reprogramming selection. Our findings provide the proof of principle that cellular reprogramming can be applied on a personalized basis to generate healthy HPCs from AML patients, and should further motivate advances toward creating transplantable hematopoietic stem cells for autologous AML therapy.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Genoma Humano , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Leucemia Mieloide Aguda/terapia , Medula Óssea/imunologia , Diferenciação Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/citologia
7.
EMBO Rep ; 15(11): 1128-38, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25252682

RESUMO

The Notch signaling pathway is evolutionarily conserved across species and plays an important role in regulating cell differentiation, proliferation, and survival. It has been implicated in several different hematopoietic processes including early hematopoietic development as well as adult hematological malignancies in humans. This review focuses on recent developments in understanding the role of Notch signaling in the human hematopoietic system with an emphasis on hematopoietic initiation from human pluripotent stem cells and regulation within the bone marrow. Based on recent insights, we summarize potential strategies for treatment of human hematological malignancies toward the concept of targeting Notch signaling for fate regulation.


Assuntos
Pleiotropia Genética , Hematopoese , Leucemia/metabolismo , Receptores Notch/genética , Transdução de Sinais , Humanos , Leucemia/genética , Receptores Notch/metabolismo
8.
Nature ; 468(7323): 521-6, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21057492

RESUMO

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Fibroblastos/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Derme/citologia , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
9.
Blood ; 121(9): 1543-52, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23293081

RESUMO

Programs that control early lineage fate decisions and transitions from embryonic to adult human cell types during development are poorly understood. Using human pluripotent stem cells (hPSCs), in the present study, we reveal reduction of Hedgehog (Hh) signaling correlates to developmental progression of hematopoiesis throughout human ontogeny. Both chemical- and gene-targeting­mediated inactivation of Hh signaling augmented hematopoietic fate and initiated transitions from embryonic to adult hematopoiesis, as measured by globin regulation in hPSCs. Inhibition of the Hh pathway resulted in truncation of Gli3 to its repressor, Gli3R, and was shown to be necessary and sufficient for initiating this transition. Our results reveal an unprecedented role for Hh signaling in the regulation of adult hematopoietic specification, thereby demonstrating the ability to modulate the default embryonic programs of hPSCs.


Assuntos
Proteínas Hedgehog/genética , Hematopoese/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Adulto , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Células Sanguíneas/metabolismo , Células Sanguíneas/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transcriptoma , Proteína Gli3 com Dedos de Zinco
10.
Blood ; 122(7): 1162-73, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23733337

RESUMO

Notch signaling regulates several cellular processes including cell fate decisions and proliferation in both invertebrates and mice. However, comparatively less is known about the role of Notch during early human development. Here, we examined the function of Notch signaling during hematopoietic lineage specification from human pluripotent stem cells of both embryonic and adult fibroblast origin. Using immobilized Notch ligands and small interfering RNA to Notch receptors we have demonstrated that Notch1, but not Notch2, activation induced hairy and enhancer of split 1 (HES1) expression and generation of committed hematopoietic progenitors. Using gain- and loss-of-function approaches, this was shown to be attributed to Notch-signaling regulation through HES1, which dictated cell fate decisions from bipotent precursors either to the endothelial or hematopoietic lineages at the clonal level. Our study reveals a previously unappreciated role for the Notch pathway during early human hematopoiesis, whereby Notch signaling via HES1 represents a toggle switch of hematopoietic vs endothelial fate specification.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Receptor Notch1/metabolismo , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Derme/citologia , Derme/metabolismo , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Células-Tronco Pluripotentes Induzidas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1
11.
Stem Cells ; 32(8): 2178-87, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24740884

RESUMO

Here we characterize the molecular and biological requirements for OCT4 plasticity induction in human skin derived fibroblasts (hFibs) that allows direct conversion of cell fate without iPSC formation. Our results indicate that adult hFibs not only require OCT4 but also short-term exposure to reprogramming media (RM) to successfully undergo direct conversion to early hematopoietic and neural progenitor fates. RM was found to be essential in this process and allowed for unique changes in global gene expression specific to the combined effects of OCT4 and treatment with reprogramming media to establish a plastic state. This molecular state of hFib plasticity was distinct from transient expression of a full complement of iPSC reprogramming factors consistent with a lack in molecular hallmarks of iPSC formation. Human Fib-derived OCT4 plastic cells display elevated levels of developmentally related genes associated with multiple lineages, but not those associated with pluripotency. In response to changes in the extracellular environment, plastic OCT4-expressing hFibs further activate genes involved in hematopoietic as well as tripotent neural progenitor biology that allow cell fate conversion. Our study provides a working definition of hFib-induced plasticity using OCT4 and a deconvoluted system to elucidate the process of direct cell fate reprogramming.


Assuntos
Linhagem da Célula , Transdiferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Fibroblastos/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco/citologia , Envelhecimento , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
12.
Curr Opin Hematol ; 21(4): 249-55, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24848771

RESUMO

PURPOSE OF REVIEW: Historically, studies of the hematopoietic stem cell (HSC) microenvironment in bone marrow have focused on the identification of individual supportive cell lineages likely to be responsible for maintaining HSCs in a self-renewing and regenerative state. More recently, awareness has developed regarding the broad and dynamic heterogeneity of nonhematopoietic cells that reside within the bone marrow space. We review recent insights that provide an emerging and complex context for understanding the spatially dependent regulation of HSC functional properties in the bone marrow and the collective inputs of multiple cell types. RECENT FINDINGS: Within the last 18 months, high-resolution imaging, xenograft modeling, and genetic mouse models have afforded innovative methods of detecting and interrogating HSCs with precision at the cellular level. Spatially distinct sites within the bone marrow house functionally divergent HSCs and progenitors, and these different habitats are becoming carefully characterized from a cellular and molecular perspective. This is critical toward understanding how bone marrow microenvironments adapt to accommodate cellular demands for hematopoiesis and how these mechanisms are disrupted in pathological conditions. SUMMARY: The bone marrow is not a continuum but an integrated unit with complex trophic interactions. Emphasis on human data will become necessary as these concepts mature and develop translationally toward changing clinical practices in HSC transplantation and even in the treatment of leukemias.


Assuntos
Células da Medula Óssea/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Diferenciação Celular , Humanos , Nicho de Células-Tronco
13.
Nat Methods ; 8(4 Suppl): S36-40, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21451511

RESUMO

Individual stem cells are functionally defined by their self-renewal and differentiation potential. Methods for clonal analysis are essential for understanding stem cells, particularly given the increasing evidence for stem-cell heterogeneity. Stem cells reside within complex microenvironments, making single-cell analysis particularly challenging. Furthermore, simultaneous molecular and functional characterization of single stem cells is not trivial. Here we explore clonal assays applied to stem cell biology and their use in understanding the cellular and molecular basis of stem-cell identity.


Assuntos
Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células Clonais/citologia , Células Clonais/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
14.
Cell Rep Med ; 5(4): 101485, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38582086

RESUMO

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells is poorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells (RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration in vivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration.


Assuntos
Perfilação da Expressão Gênica , Leucemia Mieloide Aguda , Humanos , Prognóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco/metabolismo
15.
J Cell Sci ; 124(Pt 9): 1445-52, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21486943

RESUMO

Mechanisms that govern hematopoietic lineage specification, as opposed to the expansion of committed hematopoietic progenitors, from human pluripotent stem cells (hPSCs) have yet to be fully defined. Here, we show that within the family of genes called inhibitors of differentiation (ID), ID1 and ID3 negatively regulate the transition from lineage-specified hemogenic cells to committed hematopoietic progenitors during hematopoiesis of both human embryonic stem cells (hESCs) and human induced pluripotent stem cell (hiPSCs). Upon hematopoietic induction of hPSCs, levels of ID1 and ID3 transcripts rapidly increase, peaking at the stage of hemogenic precursor emergence, and then exclusively decrease during subsequent hematopoietic commitment. Suppression of ID1 and ID3 expression in hemogenic precursors using specific small interfering RNAs augments differentiation into committed hematopoietic progenitors, with dual suppression of ID1 and ID3 further increasing hematopoietic induction compared with upon knockdown of each gene alone. This inhibitory role of ID1 and ID3 directly affects hemogenic precursors and is not dependent on non-hemogenic cells of other lineages within developing human embryoid bodies from hESCs or hiPSCs. Our study uniquely identifies ID1 and ID3 as negative regulators of the hPSC-hematopoietic transition from a hemogenic to a committed hematopoietic fate, and demonstrates that this is conserved between hESCs and hiPSCs.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Hematopoese/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Proteínas de Neoplasias/metabolismo , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Citometria de Fluxo , Hematopoese/genética , Humanos , Imuno-Histoquímica , Proteína 1 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Nat Methods ; 7(11): 917-22, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20953174

RESUMO

Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogeneous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Humanos , Fenótipo
17.
Blood ; 117(26): 7112-20, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21562049

RESUMO

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.


Assuntos
Leucemia Mieloide Aguda/patologia , Leucemia de Células T/patologia , Células-Tronco Neoplásicas/patologia , Animais , Antígenos CD34/metabolismo , Células Cultivadas , Células Clonais/metabolismo , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Sobrevivência de Enxerto , Histona-Lisina N-Metiltransferase , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , RNA Mensageiro/metabolismo , Translocação Genética , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
18.
Stem Cells ; 30(3): 392-404, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22213600

RESUMO

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N-t-hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t-hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N-hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N-t-hESCs and N-hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N-t-hESCs NPs and not from normal N-hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N-t-hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources.


Assuntos
Neoplasias Encefálicas/patologia , Células-Tronco Embrionárias/patologia , Células-Tronco Pluripotentes/patologia , Animais , Astrócitos/patologia , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica , Células Cultivadas , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Perfilação da Expressão Gênica , Humanos , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Tumores Neuroectodérmicos Primitivos/patologia , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante
19.
Stem Cells ; 30(2): 131-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22131151

RESUMO

Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here we show that, unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources.


Assuntos
Proliferação de Células , Regulação para Baixo , Hematopoese , Células-Tronco Pluripotentes Induzidas/fisiologia , MicroRNAs/genética , Regeneração , Animais , Antígenos de Diferenciação/metabolismo , Medula Óssea/fisiologia , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/metabolismo , Transplante Heterólogo
20.
Nat Med ; 12(1): 89-98, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16341242

RESUMO

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.


Assuntos
Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/fisiologia , Células-Tronco Hematopoéticas/citologia , Transplante de Células-Tronco/métodos , Animais , Glicemia/metabolismo , Proliferação de Células , Transplante de Células , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Inibidores Enzimáticos/farmacologia , Sangue Fetal/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog , Humanos , Leucócitos Mononucleares/metabolismo , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Modelos Biológicos , Neutrófilos/citologia , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco , Fatores de Tempo , Transativadores/metabolismo , Proteínas Wnt/metabolismo
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