RESUMO
The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter (low) phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Membrana Celular/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.
RESUMO
We describe in this chapter the development of a xenofree molecularly defined medium, SBX, associated with xenofree matrices, to maintain human embryonic stem cell (hESC) pluripotency as determined by phenotypic, functional and TLDA studies. This simple, inexpensive, and more physiological culture condition has been chosen because (1) it is xenofree and molecularly defined; it is devoid of albumin, which is a carrier of undefined molecules; (2) it maintains pluripotency, but very significantly reduces differentiation gene expression during hESC self-renewal, as compared to the widely used culture conditions tested so far; and (3) it can be further improved by replacing high concentrations of expensive additives by physiological concentrations of new factors. Xenofree molecularly defined media and matrices represent valuable tools for elucidating still unknown functions of numerous embryonic genes using more physiological culture conditions. These genes encode potential new factors controlling hESC self-renewal and pluripotency.
Assuntos
Meios de Cultura Livres de Soro/química , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Meios de Cultura/química , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/química , Expressão Gênica , Humanos , Células-Tronco Pluripotentes/metabolismoRESUMO
To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).
Assuntos
Células-Tronco Embrionárias/citologia , Matriz Extracelular/metabolismo , Células-Tronco Pluripotentes/citologia , Fator de Crescimento Transformador beta/farmacologia , Ativinas/farmacologia , Albuminas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Meios de Cultura , Citocinas/genética , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Cariotipagem , Fatores de Determinação Direita-Esquerda , Camundongos , Fenótipo , Fator de Crescimento Transformador beta/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Here we present a simple two-step in vitro model of vascularized trophoblastic tissue derived from human embryonic stem (hES) cells. The first step is the formation of cystic embryoid bodies (EBs) in suspension in a semisolid methyl cellulose medium, within which an endothelial platelet/endothelial cell adhesion molecule-1 (PECAM-1(+)) cell network develops. In a second step, deposition of these EBs on the bottom of nontreated, polystyrene tissue culture plates, leads by centrifugal outgrowth of the EB to the emergence of an adherent cell layer, with which a PECAM-1(+) network is associated. Cells of this adherent layer expressed VE-cadherin (CD144), PECAM-1 (CD31), and alpha-fetoprotein (alpha-FP). Trophoblastic differentiation was strongly suggested by the secretion of beta-human chorionic gonadotropin (beta-hCG) and by the presence of the cytotrophoblast and syncytiotrophoblast marker GB25. The INSL4 gene, a cyto and syncytio-trophoblast marker, was also highly expressed in the adherent layer, as well as other trophoblast genes such as CGA, CDX1, CDX2, and HAND1, compared to hES cell gene expression taken as reference. In contrast, expression of self-renewal genes, such as TERT, POU5F1, ZFP42, GDF3, and NODAL were decreased. No ectodermal or endodermal genes were expressed, but the mesodermal genes PECAM-1 and GATA2 were. The possibility of removing the EBs during the second step would permit analysis of their relative contribution to angiogenesis or possible hemangioblast formation, compared to that of the trophoblastic adherent layer. This primitive vascularized trophoblastic model could also provide a tool to study early steps of normal and pathological placental development.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Trofoblastos/fisiologia , Animais , Células Cultivadas , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Humanos , Camundongos , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Trofoblastos/citologia , alfa-Fetoproteínas/metabolismoAssuntos
Embrião de Mamíferos/citologia , Embrião não Mamífero , Expressão Gênica , Neurônios/citologia , Retina/citologia , Células-Tronco/fisiologia , Transcrição Gênica , Animais , Diferenciação Celular , Linhagem da Célula , Perfilação da Expressão Gênica , Genes , Células-Tronco Hematopoéticas/fisiologia , Integrina alfa6/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in alpha6 and beta1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-beta1, which is to promote undifferentiated keratinocyte amplification.