scholarly journals MAPK p38alpha Kinase Influences Haematopoiesis in Embryonic Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Kateřina Štefková ◽  
Markéta Hanáčková ◽  
Jan Kučera ◽  
Katarzyna Anna Radaszkiewicz ◽  
Barbora Ambrůžová ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Colony Forming Unit ◽  
Haematopoietic Cells ◽  
Colony Forming Units ◽  
First Time

The activation of p38alpha kinase mediates cell response to various extracellular factors including many interleukins and growth factors important for haematopoiesis. The role of p38alpha kinase was previously analysed in particular haematopoietic cells. In this study and for the first time, the role of p38alpha kinase in haematopoiesis was studied using a model of continuous haematopoietic development in pluripotent embryonic stem cellsin vitro. The expression of transcripts associated with haematopoiesis and the potential for the formation of specific haematopoietic cell colonies were compared between wild-type and mutant p38alpha gene-depleted cells. The absence of p38alpha kinase led to the inhibition of hemangioblast formation during the first step of haematopoiesis. Later, during differentiation, due to the lack of p38alpha kinase, erythrocyte maturation was impaired. Mutant p38α−/−cells also exhibited decreased potential with respect to the expansion of granulocyte colony-forming units. This effect was reversed in the absence of erythropoietin as shown by colony-forming unit assay in media for colony-forming unit granulocytes/macrophages. p38alpha kinase thus plays an important role in the differentiation of common myeloid precursor cells into granulocyte lineages.

scholarly journals Stimulatory Role of Glycated Fetal Bovine Serum Along with Iron on In Vitro Production of Insulin by Differentiated Mouse Embryonic Stem Cells

2011 ◽  
Vol 57 (4) ◽  
pp. 356-361
Author(s):  
Ikuo Nishigaki ◽  
Gowri Rangasamy Gunassekaran ◽  
Panjan Nagappan Venkatesan ◽  
Mandupal Chaco Sabu ◽  
Sabu Priya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Embryonic Stem ◽  
In Vitro Production ◽  
Fetal Bovine ◽  
Vitro Production

Role of Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mice in vitro

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 513-520 ◽  
Author(s):  
I. Bahena ◽  
E. Xu ◽  
M. Betancourt ◽  
E. Casas ◽  
Y. Ducolomb ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Embryonic Stem ◽  
Oocyte Growth ◽  
Germ Cell Differentiation ◽  
Rnai Technology

SummaryIn a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.

scholarly journals Absence of Rybp Compromises Neural Differentiation of Embryonic Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Gergo Kovacs ◽  
Viktoria Szabo ◽  
Melinda K. Pirity
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Wild Type ◽  
Altered Gene Expression ◽  
Molecular Events ◽  
Altered Gene ◽  
Polycomb Repressive Complex 1

Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp+/+) andrybp nullmutant (rybp-/-) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found thatrybp nullmutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack ofrybpcoincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes.

scholarly journals Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

2019 ◽  
Vol 20 (5) ◽  
pp. 1236 ◽  
Author(s):  
Wei-Fang Chang ◽  
Yun-Hsin Wu ◽  
Jie Xu ◽  
Li-Ying Sung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Population Doubling ◽  
Chondrocyte Differentiation ◽  
Wild Type

Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (TERC). We previously reported the generation of mouse Terc+/− and Terc−/− embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of Terc−/−, Terc+/− and wild-type (Terc+/+) ntESCs. The telomere lengths are longest in wild-type but shortest in Terc−/− ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that Terc−/− ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the Terc−/− cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.

scholarly journals The Role of Sequential BMP Signaling in Directing Human Embryonic Stem Cells to Bipotential Gonadal Cells

2017 ◽  
Vol 102 (11) ◽  
pp. 4303-4314 ◽  
Author(s):  
Kirsi Sepponen ◽  
Karolina Lundin ◽  
Katri Knuus ◽  
Pia Väyrynen ◽  
Taneli Raivio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Academic Research ◽  
Embryonic Stem ◽  
Gonadal Development ◽  
Activin A ◽  
Bmp Signaling

Abstract Context Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed. Objective To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation. Design We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation. Setting Academic research laboratory. Main Outcomes Measures Cell differentiation, gene expression, and flow cytometry. Results The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters. Conclusions We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.

scholarly journals THE ROLE OF SUBSTRATE STIFFNESS IN MAINTAINING PLURIPOTENCY OF EMBRYONIC STEM CELLS IN VITRO CULTURE

2021 ◽  
Vol 67 (3) ◽  
pp. 27-34
Author(s):  
D.I. Bilko ◽  
◽  
Y.B. Chaikovsky ◽  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Embryonic Stem Cells ◽  
In Vitro Culture ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Polyacrylamide Gel ◽  
Substrate Stiffness

Murine embryonic stem cells (ESCm) cultured in vitro in the presence of LIF (leukemia inhibitory factor) maintain pluripotency. However, when LIF is removed from the media, an active differentiation into various specialized somatic cells is observed. The aim of the study was to determine the role of substrate stiffness in maintaining of pluripotency of embryonic stem cells in vitro culture. To this aim, we used the method of culturing pluripotent stem cells in vitro, the method of “hanging drop”, the determination of the Young’s modulus for polyacrylamide gel of different hardness, the immunocytochemical alkaline phosphatase (AP) streptavidin-biotin method, microscopy. By culturing ESCm on a soft, medium and hard density polyacrylamide gel as a substrate (0.8, 4.0, 8.0 кPа), we found that on a soft gel ESCm differentiation does not occur even in the absence of LIF. ESCm cultured on a soft substrate continue to show signs of pluripotency, namely, create round compact colonies with high alkaline phosphatase activity and form embryoid bodies (EB), the efficiency of which (87.5 ± 3.2 per 100 cells seeded) did not decrease even after LIF withdrawal. In the absence of LIF, ESCs cultured on a hard base showed a low level of EB formation (23.5 ± 2.24). The results of our observations demonstrate that the process of EB formation may be influenced not only by a composition of nutrient medium, but also by complex interaction between the physical forces of the matrix and the mechanical properties of 3D cell aggregates. The model is considered as a tool to study early events in embryogenesis in the search of conditions for effective culture of progenitor cells and differentiated cells for transplantation.

scholarly journals Hematopoietic-Specific Genes Are Not Induced During In Vitro Differentiation of scl-Null Embryonic Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1435-1447 ◽  
Author(s):  
Andrew G. Elefanty ◽  
Lorraine Robb ◽  
Raquella Birner ◽  
C. Glenn Begley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Embryoid Bodies ◽  
Globin Genes ◽  
Wild Type ◽  
Temporal Expression Pattern

Abstract The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation.

scholarly journals Hematopoietic-Specific Genes Are Not Induced During In Vitro Differentiation of scl-Null Embryonic Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1435-1447 ◽  
Author(s):  
Andrew G. Elefanty ◽  
Lorraine Robb ◽  
Raquella Birner ◽  
C. Glenn Begley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Embryoid Bodies ◽  
Globin Genes ◽  
Wild Type ◽  
Temporal Expression Pattern

The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation.

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