RESUMO
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
Assuntos
Pesquisa com Células-Tronco , Humanos , Reprodutibilidade dos TestesRESUMO
Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of putative stem cell origin that predominantly occurs in children and young adults. Although most patients with localized ES can be cured with intensive therapy, the clinical course is variable and up to one third of patients relapse following initial remission. Unfortunately, little is yet known about the biologic features that distinguish low-risk from high-risk disease or the mechanisms of ES disease progression. Recent reports have suggested that putative cancer stem cells exist in ES and may contribute to an aggressive phenotype. The cell surface receptor leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a somatic stem cell marker that functions as an oncogene in several human cancers, most notably colorectal carcinoma. LGR5 is a receptor for the R-spondin (RSPO) family of ligands and RSPO-mediated activation of LGR5 potentiates Wnt/ß-catenin signaling, contributing to stem cell proliferation and self-renewal. Given its presumed stem cell origin, we investigated whether LGR5 contributes to ES pathogenesis. We found that LGR5 is expressed by ES and that its expression is relatively increased in cells and tumors that display a more aggressive phenotype. In particular, LGR5 expression was increased in putative cancer stem cells. We also found that neural crest-derived stem cells express LGR5, raising the possibility that expression of LGR5 may be a feature of ES cells of origin. LGR5-high ES cells showed nuclear localization of ß-catenin and robust activation of TCF reporter activity when exposed to Wnt ligand and this was potentiated by RSPO. However, modulation of LGR5 or exposure to RSPO had no impact on proliferation confirming that Wnt/ß-catenin signaling in ES cells does not recapitulate signaling in epithelial cells. Together these studies show that the RSPO-LGR5-Wnt-ß-catenin axis is present and active in ES and may contribute to tumor pathogenesis.
RESUMO
Abnormalities in the development of enteric neural crest-derived progenitors (ENPs) that generate the enteric nervous system (ENS) can lead to aganglionosis in a variable portion of the distal gastrointestinal tract. Cumulative evidence suggests that variation of aganglionosis is due to gene interactions that modulate the ability of ENPs to populate the intestine; however, the developmental processes underlying this effect are unknown. We hypothesized that differences in enteric ganglion deficits could be attributable to the effects of genetic background on early developmental processes, including migration, proliferation, or lineage divergence. Developmental processes were investigated in congenic Sox10(Dom) mice, an established Hirschsprung disease (HSCR) model, on distinct inbred backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). Immuno-staining on whole-mount fetal gut tissue and dissociated cell suspensions was used to assess migration and proliferation. Flow cytometry utilizing the cell surface markers p75 and HNK-1 was used to isolate live ENPs for analysis of developmental potential. Frequency of ENPs was reduced in Sox10(Dom) embryos relative to wild-type embryos, but was unaffected by genetic background. Both migration and developmental potential of ENPs in Sox10(Dom) embryos were altered by inbred strain background with the most highly significant differences seen for developmental potential between strains and genotypes. In vivo imaging of fetal ENPs and postnatal ganglia demonstrates that altered lineage divergence impacts ganglia in the proximal intestine. Our analysis demonstrates that genetic background alters early ENS development and suggests that abnormalities in lineage diversification can shift the proportions of ENP populations and thus may contribute to ENS deficiencies in vivo.
Assuntos
Sistema Nervoso Entérico/embriologia , Doença de Hirschsprung/genética , Crista Neural/citologia , Fatores de Transcrição SOXE/genética , Células-Tronco/citologia , Animais , Antígenos CD57/metabolismo , Modelos Animais de Doenças , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Gânglios/embriologia , Gânglios/patologia , Doença de Hirschsprung/embriologia , Doença de Hirschsprung/metabolismo , Humanos , Imuno-Histoquímica , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestinos/citologia , Intestinos/embriologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação , Crista Neural/embriologia , Especificidade da EspécieRESUMO
Neurofibromatosis is caused by the loss of neurofibromin (Nf1), leading to peripheral nervous system (PNS) tumors, including neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). A long-standing question has been whether these tumors arise from neural crest stem cells (NCSCs) or differentiated glia. Germline or conditional Nf1 deficiency caused a transient increase in NCSC frequency and self-renewal in most regions of the fetal PNS. However, Nf1-deficient NCSCs did not persist postnatally in regions of the PNS that developed tumors and could not form tumors upon transplantation into adult nerves. Adult P0a-Cre+Nf1(fl/-) mice developed neurofibromas, and Nf1(+/-)Ink4a/Arf(-/-) and Nf1/p53(+/-) mice developed MPNSTs, but NCSCs did not persist postnatally in affected locations in these mice. Tumors appeared to arise from differentiated glia, not NCSCs.
Assuntos
Neoplasias/patologia , Crista Neural/citologia , Neurofibromina 1/deficiência , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Mutação/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Neoplasias de Bainha Neural/patologia , Crista Neural/efeitos dos fármacos , Neurofibroma Plexiforme/patologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/embriologia , Sistema Nervoso Periférico/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/metabolismoRESUMO
We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Embrionárias/fisiologia , Sistema Nervoso Entérico/embriologia , Doença de Hirschsprung/terapia , Crista Neural/citologia , Animais , Células Cultivadas , Embrião de Galinha , Primers do DNA , Células-Tronco Embrionárias/citologia , Sistema Nervoso Entérico/citologia , Feminino , Trato Gastrointestinal/citologia , Imuno-Histoquímica , Hibridização In Situ , Melanócitos/citologia , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Endotelina/metabolismo , Nervo Isquiático/citologia , Transplante de Células-TroncoRESUMO
Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.
Assuntos
Diferenciação Celular , Linhagem da Célula , Endotélio/citologia , Crista Neural/citologia , Crista Neural/embriologia , Células de Schwann/citologia , Células-Tronco/citologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Modelos Biológicos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Neuregulina-1/metabolismo , Fenótipo , Ratos , Receptores Fc/metabolismo , Nervo Isquiático/citologia , Antígenos Thy-1/metabolismoRESUMO
Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.
Assuntos
Movimento Celular/fisiologia , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Receptor de Endotelina B/fisiologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Feminino , Trato Gastrointestinal/citologia , Humanos , Crista Neural/citologia , Gravidez , Ratos , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Receptor de Endotelina B/deficiência , Receptor de Endotelina B/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologiaRESUMO
Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Sistema Nervoso Entérico/embriologia , Crista Neural/embriologia , Neuroglia/metabolismo , Neurônios/metabolismo , Nervo Isquiático/embriologia , Células-Tronco/metabolismo , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem da Célula/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Feminino , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Fatores de Crescimento Neural/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Neuregulina-1/metabolismo , Neuregulina-1/farmacologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.