RESUMO
Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.
Assuntos
Diabetes Mellitus Tipo 1 , Células-Tronco Embrionárias Humanas , Animais , Diferenciação Celular , Humanos , Mesoderma , CamundongosRESUMO
Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.
Assuntos
Células-Tronco Embrionárias/metabolismo , Mutação/genética , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Transcrição Gênica , Ativinas/genética , Ativinas/metabolismo , Animais , Sequência de Bases , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Engenharia Genética , Loci Gênicos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas/genética , RNA não Traduzido , Ratos , Receptores Notch/genética , Receptores Notch/metabolismo , Recombinação Genética/genética , Elementos de Resposta/genética , Deleção de Sequência/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Embryonic stem (ES) cells differentiating as aggregates self-organize dependent on Wnt signaling that is initially localized to discrete sites in the aggregate. As differentiation proceeds, Wnt signaling expands to most of the aggregates, thus resulting in widespread differentiation of mesendodermal progenitors. This process resembles primitive streak formation, but the lack of organized positional information makes the differentiating aggregates develop in a disorganized fashion. Here, we report that exogenous, cellular signaling sources can control the site where differentiation initiates in ES cell aggregates. Fibroblasts engineered to express cadherins are assembled with ES cells to form composite aggregates where the fibroblasts are positioned as a discrete pole. When engineered to express secreted Wnt agonists or antagonists, this pole functions to localize signaling in a way that polarizes the differentiating aggregates. The use of cell adhesion molecules to control morphology of developing stem cell aggregates should be widely applicable in tissue engineering.
