RESUMO
In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Linhagem da Célula/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Fenômenos Biomecânicos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/genética , Gastrulação/efeitos dos fármacos , Gastrulação/genética , Perfilação da Expressão Gênica , Heterogeneidade Genética , Ensaios de Triagem em Larga Escala , Humanos , Modelos Biológicos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Proteína Nodal/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Propriedades de SuperfícieRESUMO
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3ß and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , TranscriptomaRESUMO
It is well known that human embryonic stem cells (hESCs) frequently acquire recurrent chromosomal abnormalities, very reminiscent of those found in cancerous cells. Given the parallels between cancer and stem cell biology, we set out to investigate the occurrence of a common form of genome instability in tumors, namely microsatellite instability (MSI), in hESCs. MSI is caused by a deficiency in mismatch repair (MMR) genes, which leads to the accumulation of mutations during DNA replication. In this study, we analyzed up to 122 microsatellites in a total of 10 hESC lines, for 1-11 different passages, ranging from passage 7 to passage 334. In two lines, this revealed that two microsatellites had altered allelic patterns. Small-pool PCR for several microsatellites and testing of the Bethesda panel microsatellites (commonly used in cancer studies) revealed that, whilst MSI is common in all tested lines, it occurs at a very low and variable frequency, ranging from â¼1 to 20% of the total number of alleles. In cancerous cells, MSI leads to multiple large shifts in allele sizes within the majority of the cells, while hESCs show small changes in a minority of the cells. Since these genetic alterations do not consistently take over the culture, we assume that they are not concurrent with a selective advantage as it is in tumors. Finally, the MMR genes showed a very variable gene expression that could not be correlated with the variable (low) levels of MSI in the different hESC lines.
Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Instabilidade de Microssatélites , Linhagem Celular , Hibridização Genômica Comparativa , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Humanos , Taxa de MutaçãoRESUMO
Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.
Assuntos
Ativinas/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Endoderma/citologia , Fatores de Crescimento de Fibroblastos/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Contagem de Células , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Pâncreas/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
PURPOSE: To investigate the spontaneous germ cell differentiation capacity of VUB hESC lines, develop a protocol for the induction of germ cell differentiation using conditioned medium from Sertoli cells (SCCM) and compare it to existing protocols. METHODS: hESC were allowed to differentiate spontaneously or after the addition of bone morphogenetic proteins (BMPs) and/or SCCM. VASA transcripts were measured by relative quantification real-time RT-PCR to determine the efficiency of germ cell differentiation. RESULTS: VUB hESC lines can differentiate spontaneously towards the germ cell lineage, however, more consistently in an embryoid body approach than in monolayer cultures. BMPs and SCCM significantly improve VASA expression, but do not have a synergistic effect. Direct contact of differentiating hESC with Sertoli cells does not improve VASA expression. CONCLUSIONS: SCCM contains inductive factors for germ cell differentiation and could represent an element for in-vitro differentiation to germ cells.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Células de Sertoli/citologia , Animais , Linhagem da Célula , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/metabolismo , Células Germinativas/metabolismo , Humanos , Masculino , Camundongos , Células de Sertoli/metabolismoRESUMO
Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.
Assuntos
Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas , Via de Sinalização Wnt , Diferenciação Celular , Linhagem Celular , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , HumanosRESUMO
BACKGROUND: The aim of this study was to explore the plasticity and transdifferentiation potential of murine spermatogonial stem cells (SSCs) into hematopoietic cells. METHODS: GFP(+)CD49f(+)H-2K(b-) SSCs of male donor mice were isolated and injected into the bone marrow (BM) of Busulfan-treated GFP(-) female mice. Twelve weeks post-transplantation, the recipients were sacrificed and their BM, peripheral blood (PB) and spleen (SL) cells were collected and evaluated by phenotypical methods, i.e. fluorescence-activated cell sorting, immunohistochemistry and fluorescence in situ hybridization, and functional assays, i.e. colony-forming units assay and intra-BM transplantation. RESULTS: Green fluorescent protein (GFP)- and Y chromosome-positive cells were observed in the BM, PB and SL of transplanted female mice. These cells presented phenotypical and functional characteristics of hematopoietic cells in vitro and in vivo. CONCLUSIONS: Our results indicate that SSCs have the potential to transdifferentiate into hematopoietic cells in vivo.
Assuntos
Transdiferenciação Celular , Células-Tronco Hematopoéticas/fisiologia , Espermatogônias/citologia , Células-Tronco/fisiologia , Animais , Feminino , Masculino , CamundongosRESUMO
BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.
Assuntos
Blastômeros/citologia , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias , Linhagem Celular , Desenvolvimento Embrionário , Humanos , Células-Tronco PluripotentesRESUMO
Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.
Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Embrionárias Humanas , Mosaicismo , Análise de Célula Única , Linhagem Celular , HumanosRESUMO
BACKGROUND: Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. SEARCH METHODS: Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. OUTCOMES: Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. WIDER IMPLICATIONS: As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.
RESUMO
In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.
Assuntos
Diferenciação Celular/genética , Evolução Clonal/genética , DNA Mitocondrial , Mutagênese , Células-Tronco Pluripotentes/metabolismo , Alelos , Técnicas de Cultura de Células , Aberrações Cromossômicas , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Heterogeneidade Genética , Variação Genética , Instabilidade Genômica , Genótipo , Humanos , MosaicismoRESUMO
BACKGROUND: Human pluripotent stem cells (hPSC), both embryonic and induced (hESC and hiPSC), are regarded as a valuable in vitro model for early human development. In order to fulfil this promise, it is important that these cells mimic as closely as possible the in vivo molecular events, both at the genetic and epigenetic level. One of the most important epigenetic events during early human development is X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female cells. XCI is important for proper development and aberrant XCI has been linked to several pathologies. Recently, novel data obtained using high throughput single-cell technology during human preimplantation development have suggested that the XCI mechanism is substantially different from XCI in mouse. It has also been suggested that hPSC show higher complexity in XCI than the mouse. Here we compare the available recent data to understand whether XCI during human preimplantation can be properly recapitulated using hPSC. OBJECTIVE AND RATIONALE: We will summarize what is known on the timing and mechanisms of XCI during human preimplantation development. We will compare this to the XCI patterns that are observed during hPSC derivation, culture and differentiation, and comment on the cause of the aberrant XCI patterns observed in hPSC. Finally, we will discuss the implications of the aberrant XCI patterns on the applicability of hPSC as an in vitro model for human development and as cell source for regenerative medicine. SEARCH METHODS: Combinations of the following keywords were applied as search criteria in the PubMed database: X chromosome inactivation, preimplantation development, embryonic stem cells, induced pluripotent stem cells, primordial germ cells, differentiation. OUTCOMES: Recent single-cell RNASeq data have shed new light on the XCI process during human preimplantation development. These indicate a gradual inactivation on both XX chromosomes, starting from Day 4 of development and followed by a random choice to inactivate one of them, instead of the mechanism in mice where imprinted XCI is followed by random XCI. We have put these new findings in perspective using previous data obtained in human (and mouse) embryos. In addition, there is an ongoing discussion whether or not hPSC lines show X chromosome reactivation upon derivation, mimicking the earliest embryonic cells, and the XCI states observed during culture of hPSC are highly variable. Recent studies have shown that hPSC rapidly progress to highly aberrant XCI patterns and that this process is probably driven by suboptimal culture conditions. Importantly, these aberrant XCI states seem to be inherited by the differentiated hPSC-progeny. WIDER IMPLICATIONS: The aberrant XCI states (and epigenetic instability) observed in hPSC throw a shadow on their applicability as an in vitro model for development and disease modelling. Moreover, as the aberrant XCI states observed in hPSC seem to shift to a more malignant phenotype, this may also have important consequences for the safety aspect of using hPSC in the clinic.
Assuntos
Cromossomos Humanos X , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas/fisiologia , Inativação do Cromossomo X , Animais , Diferenciação Celular , Feminino , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , RNA Longo não Codificante/metabolismoRESUMO
When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.
Assuntos
Proliferação de Células/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Laminina/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.
Assuntos
Técnicas de Cultura de Células/métodos , Dano ao DNA , Células-Tronco Embrionárias/citologia , Instabilidade Genômica , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Laminina/farmacologiaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is associated with an activation of the double homeobox 4 (DUX4) gene, which we previously identified within the D4Z4 repeated elements in the 4q35 subtelomeric region. The pathological DUX4 mRNA is derived from the most distal D4Z4 unit and extends unexpectedly within the flanking pLAM region, which provides an intron and polyadenylation signal. The conditions that are required to develop FSHD are a permissive allele providing the polyadenylation signal and hypomethylation of the D4Z4 repeat array compared with the healthy muscle. The DUX4 protein is a 52-kDa transcription factor that initiates a large gene deregulation cascade leading to muscle atrophy, inflammation, differentiation defects, and oxidative stress, which are the key features of FSHD. DUX4 is a retrogene that is normally expressed in germline cells and is submitted to repeat-induced silencing in adult tissues. Since DUX4 mRNAs have been detected in human embryonic and induced pluripotent stem cells, we investigated whether they could also be expressed in human mesenchymal stromal cells (hMSCs). We found that DUX4 mRNAs were induced during the differentiation of hMSCs into osteoblasts and that this process involved DUX4 and new longer protein forms (58 and 70 kDa). A DUX4 mRNA with a more distant 5' start site was characterized that presented a 60-codon reading frame extension and encoded the 58-kDa protein. Transfections of hMSCs with an antisense oligonucleotide targeting DUX4 mRNAs decreased both the 52- and 58-kDa protein levels and confirmed their identity. Gain- and loss-of-function experiments in hMSCs suggested these DUX4 proteins had opposite roles in osteogenic differentiation as evidenced by the alkaline phosphatase activity and calcium deposition. Differentiation was delayed by the 58-kDa DUX4 expression and it was increased by 52-kDa DUX4. These data indicate a role for DUX4 protein forms in the osteogenic differentiation of hMSCs.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/citologia , Osteogênese , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Current knowledge on chromosomal mosaicism in human cell cultures is mostly based on cytogenetic banding methods. The recent development of high-resolution full-genome analysis methods applicable to single cells is providing new insights into genetic and cellular diversity. Here we study the genetic content of 92 individual human cells, including fibroblasts, amniocytes and embryonic stem cells (hESCs), using single-cell array-based comparative genomic hybridization (aCGH). We find that human somatic and embryonic stem cell cultures show significant fractions of cells carrying unique megabase-scale chromosomal abnormalities, forming genetic mosaics that could not have been detected by conventional cytogenetic methods. These findings are confirmed by studying seven clonal hESC sub-lines by aCGH. Furthermore, fluorescent in situ hybridisation reveals an increased instability of the subtelomeric regions in hESC as compared to somatic cells. This genetic heterogeneity may have an impact on experimental results and, in the case of hESC, on their potential clinical use.
Assuntos
Âmnio/metabolismo , Cromossomos Humanos/genética , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Mosaicismo , Âmnio/citologia , Linhagem Celular , Células Cultivadas , Hibridização Genômica Comparativa , Humanos , Hibridização in Situ FluorescenteRESUMO
Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Sinaptofisina/genética , Sinaptofisina/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transplante HeterólogoRESUMO
More than 600 human embryonic stem cell (hESC) lines have been reported today at the human European Embryonic Stem Cell Registry ( http://www.hescreg.eu/ ). Despite these high numbers, there are currently no general protocols for derivation, culture, and characterization of hESC. Moreover, data on the culture of the embryo used for the derivation (medium, day of ICM isolation) are usually not available but can have an impact on the derivation rate. We present here the protocols for derivation, culture and characterization as we applied them for the 22 hESC lines (named VUB-hESC) in our laboratory.
Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Linhagem Celular , HumanosRESUMO
OBJECTIVE: To investigate whether a selective matrix adhesion-based protocol can enrich germ cells and deplete cancer cells from contaminated human testicular cell suspensions. DESIGN: Artificially contaminated and control testicular cell suspensions were selectively cultured. After each experimental step, the presence of B cells was evaluated by fluorescence-activated cell sorting (FACS) and/or polymerase chain reaction (PCR). SETTING: Academic medical center. PATIENT(S): Five vasectomy reversal patients willing to donate testicular tissue for research after informed consent. INTERVENTION(S): In vitro culture, magnetic-activated cell sorting (MACS), matrix adhesion, FACS, and PCR. MAIN OUTCOME MEASURE(S): FACS analysis for CD49f and human leukocyte antigen (HLA) class I expression; PCR analysis for a 120-bp fragment of the B-cell receptor. RESULT(S): With the use of a matrix adhesion-based protocol, CD49f(+) HLA class I(-) cells could be highly enriched, but HLA class I(+) CD49f(-) cells could still be detected. PCR proved that the cell suspensions contained malignant cells originating from the cell line used to contaminate them. CONCLUSION(S): Even though an efficient enrichment of germ cells was achieved, the resulting population was not pure. Malignant cells were detected in selected cell suspensions of all five patients, demonstrating that the efficacy of this protocol is insufficient for clinical application.
Assuntos
Separação Celular/métodos , Neoplasias Testiculares/patologia , Testículo/patologia , Algoritmos , Adesão Celular , Matriz Extracelular/metabolismo , Citometria de Fluxo/métodos , Células Germinativas/metabolismo , Células Germinativas/patologia , Células Germinativas/fisiologia , Humanos , Masculino , Especificidade por Substrato , Suspensões , Testículo/químicaRESUMO
Although childhood cancer treatments are yielding higher survival rates, sterility remains one of the major side effects. For prepubertal boys there are currently no options to preserve fertility. Testicular tissue banking together with subsequent grafting may become a possible strategy in the future. In the present study, we compared two cryopreservation protocols using prepubertal murine testicular tissue. Fresh and cryopreserved testicular tissue was grafted subcutaneously on the back of immune-deficient mice for at least 3 months. Prepubertal murine tissue recovered well after cryopreservation with both ethylene glycol (EG) and dimethylsulfoxide (DMSO). While in fresh murine allografts, spermatozoa were observed in 23% of the tubules; in both the DMSO and the EG groups, 32% of the seminiferous tubules contained spermatozoa. However, with DMSO the structure of the seminiferous tubules was better preserved.