scholarly journals Reversible programming of pluripotent cell differentiation

2000 ◽  
Vol 113 (3) ◽  
pp. 555-566 ◽  
Author(s):  
J. Lake ◽  
J. Rathjen ◽  
J. Remiszewski ◽  
P.D. Rathjen
Keyword(s):  
Gene Expression ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Primitive Endoderm ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Differentiation Potential

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types

2001 ◽  
Vol 13 (1) ◽  
pp. 15 ◽  
Author(s):  
J. Rathjen ◽  
S. Dunn ◽  
M. D. Bettess ◽  
P. D. Rathjen
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Cell Aggregates ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Pluripotent Cells ◽  
Developmental Potential

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

scholarly journals Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hye-Yeong Jo ◽  
Youngsun Lee ◽  
Hongryul Ahn ◽  
Hyeong-Jun Han ◽  
Ara Kwon ◽  
...  
Keyword(s):  
Cell Fate ◽  
Early Stage ◽  
Expression Patterns ◽  
Transcriptome Profiling ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Negative Effect ◽  
In Vitro Effects

Abstract Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 535 ◽  
Author(s):  
T. A. Pelton ◽  
M. D. Bettess ◽  
J. Lake ◽  
J. Rathjen ◽  
P. D. Rathjen
Keyword(s):  
Cell Biology ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Experimental Manipulation ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Cellular Origin

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.

182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 215
Author(s):  
M. Nowak-Imialek ◽  
X. Gao ◽  
P. Liu ◽  
H. Niemann
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

215 COMPARATIVE ANALYSIS OF PORCINE PLURIPOTENT CELL LINES DERIVED FROM SOMATIC CELLS AND VARIOUS EMBRYONIC ORIGINS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. K. Park ◽  
H. S. Kim ◽  
K. J. Uh ◽  
K. H. Choi ◽  
H. M. Kim ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Pluripotent Cells ◽  
Differentiation Potential

Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts, tremendous efforts have been made to establish embryonic stem cell (ESC) lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to derive pluripotent cells of naïve state that represents full pluripotency, due to the frequent occurrence of spontaneous differentiation into an EpiSC-like state during culture in pigs. We have been able to derive EpiSC-like porcine embryonic stem cell (pESC) lines of a differentiated non-ES cell state from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. In this study, we analysed characteristics such as marker expression, pluripotency and the X chromosome inactivation (XCI) status of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential; female XCI activity and a normal karyotype. Here we provide preliminary results that suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. This work was supported by the BioGreen 21 Program (#20070401034031, PJ0081382011), Rural Development Administration, Republic of Korea.

scholarly journals Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Tielens ◽  
B Verhasselt ◽  
J Liu ◽  
M Dhont ◽  
J Van Der Elst ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Stem Cell Lines

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Sincein vitroculture may influence Oct-4 expression, we investigated to what extent blastocysts culturedin vitrofrom the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We comparedin vivowithin vitroderived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells’ differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of thein vitroderived blastocysts, 17% gave rise to ES cells vs 38% of thein vivoblastocysts. Six-day old outgrowths fromin vivodeveloped blastocysts expressed Oct-4 in 55% of the cases vs 31% of thein vitroderived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collectedin vivoblastocysts compared toin vitrocultured blastocysts.In vitrocultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level thanin vivoblastocysts.

HOXB4 Engages in Signaling Pathways Associated with HSC Self Renewal.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1344-1344
Author(s):  
Bernhard Schiedlmeier ◽  
Ana Santos ◽  
Hannes Klump ◽  
Ana Ribeiro ◽  
Herbert Auer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Signaling Pathways ◽  
Target Genes ◽  
Embryoid Bodies ◽  
Gene Products ◽  
Tnf Receptor ◽  
Self Renewal

Abstract Ectopic expression of the transcription factor HOXB4 has been shown to mediate expansion of adult hematopoietic stem cells (HSCs) in vitro and in vivo, and to generate embryonic stem cell (ESC) derived HSCs capable to reconstitute lethally irradiated mice. To identify target genes of HOXB4 in adult HSCs and progenitor cells proliferating in vitro, we transduced murine cells with a retroviral construct that coexpresses EGFP and an inducible form of HOXB4 (HOXB4ER). Upon addition of 4-hydroxytamoxifen (TMX), HOXB4ER translocates from the cytoplasma to the nucleus, thereby being capable to modulate gene expression. Transduced cell populations were expanded for 14 days in the presense of TMX. Gene expression profiles were obtained from FACS-sorted HOXB4ER+ LSK (lineage neagtive, Sca1+, ckit+ ) cells after culture with and without TMX. As a control, profiling was performed with LSK cells expressing unmodified active HOXB4 ± TMX. On the Genechip Mouse Genome 430 2.0 Array (Affymetrix) we observed 110 characterized gene products to be differentially expressed 4 hours after inactivation of HOXB4. Globally, the Gene Ontology categories “signal transduction”, “intracellular signaling cascade”, “Wnt receptor signaling” and “cell differentiation” were significantly over-represented among the group of up-regulated HOXB4 target genes. The list of down-regulated genes revealed a significant overrepresentation of the GO categories “regulation of cell cycle”, “regulation of apoptosis” and “regulation of DNA-dependent transcription”. Importantly, differential expression of genes involved in several signaling pathways known to either stimulate or inhibit HSC self renewal (WNT, Notch, FGF, TGFβ/BMP and TNFα) was observed as well as a high degree of concordance with HSC-specific genes discovered in previous microarray reports. TNF receptor 2 was down-regulated by HOXB4 and an inhibitor of TNF receptor 1, BRE (brain and reproductive organ expressed protein) was up-regulated. This suggested that HOXB4-expressing HSCs may still be capable of undergoing self renewal cell divisions in vitro, even in the presence of TNFα. Thus, LSK cells were transduced with the inducible HOXB4ER vector or with a control vector expressing a truncated form of human CD34 (tCD34), pooled in a 1:1 ratio, and cultivated for 7 days in serum-free medium with and without TNFα ± TMX. In the absence of TNFα and TMX, in vitro cultured HOXB4ER+ as well as tCD34+ cells both maintained high levels of multilineage reconstitution activity in vivo. However, in the presence of TNFα without HOXB4 induction, the ability of reconstitution was almost completely lost. In contrast, in the presence of TNFα, high levels of multilineage reconstitution were achieved with TMX- induced HOXB4ER cells, but not so with tCD34+ control cells. Hence, HOXB4 renders long-term repopulating HSCs insensitive to the negative effects of TNFα. We also analyzed gene expression changes in a murine ES cells containing a tetracycline-inducible HOXB4 gene. Over 700 characterized genes were differentially regulated 48 hours after doxycycline-mediated HOXB4 induction in day 6 embryoid bodies (EB). Remarkably, HOXB4 engaged in the same aforementioned signal transduction pathways during EB differentiation, in part by targeting identical gene products. In summary, our data and functional studies reveal that HOXB4 changes the response of ESC and HSC to extrinsic cues regulating self renewal and differentiation.

scholarly journals Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

2002 ◽  
Vol 115 (2) ◽  
pp. 329-339 ◽  
Author(s):  
T. A. Pelton ◽  
S. Sharma ◽  
T. C. Schulz ◽  
J. Rathjen ◽  
P. D. Rathjen
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Indirect Evidence ◽  
Cell Populations ◽  
Molecular Evidence ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Cell Progression

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4+ pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.

scholarly journals Polycomb enables primitive endoderm lineage priming in embryonic stem cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Robert S Illingworth ◽  
Jurriaan J Hölzenspies ◽  
Fabian V Roske ◽  
Wendy A Bickmore ◽  
Joshua M Brickman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Developmental Trajectory ◽  
Primitive Endoderm ◽  
Dynamic Changes ◽  
Lineage Priming

Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene’s developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision.

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