scholarly journals Scalable in vitro production of defined mouse erythroblasts

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261950
Author(s):  
Helena S. Francis ◽  
Caroline L. Harold ◽  
Robert A. Beagrie ◽  
Andrew J. King ◽  
Matthew E. Gosden ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Primary Cells ◽  
In Vitro Production ◽  
Lineage Specification ◽  
Multipotent Cells ◽  
Epigenetic Analysis ◽  
Primitive Erythropoiesis ◽  
Vitro Production

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

scholarly journals Scalable In Vitro Production of Defined Mouse Erythroblasts

2020 ◽  
Author(s):  
Helena S Francis ◽  
Caroline L Harold ◽  
Robert A Beagrie ◽  
Andrew J King ◽  
Matthew E Gosden ◽  
...  
Keyword(s):  
Mouse Models ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
List Type ◽  
Primary Cells ◽  
Erythroid Cells ◽  
In Vitro Production ◽  
Primitive Erythropoiesis ◽  
Vitro Production

AbstractMouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.Key PointsScalable purification of primitive-like erythroid cells from in vitro differentiated mESCs offers tractable tools for genetic studiesIn vitro derived erythroid cells recapitulate wild type and engineered mutation phenotypes observed in primary cells obtained from mouse models

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

scholarly journals Gelatin Electrospun Mat as a Potential Co-culture System for In Vitro Production of Sperm Cells from Embryonic Stem Cells

2020 ◽  
Vol 6 (10) ◽  
pp. 5823-5832
Author(s):  
Mina Vardiani ◽  
Marefat Ghaffari Novin ◽  
Morteza Koruji ◽  
Hamid Nazarian ◽  
Ellen Goossens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Sperm Cells ◽  
In Vitro Production ◽  
Vitro Production

scholarly journals Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells

2013 ◽  
Vol 305 (3) ◽  
pp. E325-E335 ◽  
Author(s):  
Jixiu Shan ◽  
Takashi Hamazaki ◽  
Tiffany A. Tang ◽  
Naohiro Terada ◽  
Michael S. Kilberg
Keyword(s):  
Stem Cells ◽  
Amino Acid ◽  
Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Cell ◽  
Lineage Specification ◽  
Amino Acid Response

In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages.

262. Towards derivation of primordial germ cells from murine embryonic stem cells

2005 ◽  
Vol 17 (9) ◽  
pp. 106
Author(s):  
J. C. Young ◽  
V. L. Dias ◽  
M. Holland ◽  
K. L. Loveland
Keyword(s):  
Culture Media ◽  
Es Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Lineage Specification ◽  
Signalling Molecules

The process of primordial germ cell (PGC) specification begins at the earliest stages of murine embryogenesis. The mechanisms underlying this process are the strong instructive cues generated by the extra-embryonic ectoderm, which, via ligand-receptor signalling in the visceral endoderm, activate pathways in the proximal epiblast to induce the PGC phenotype. We have subjected murine embryonic stem (ES) cells to similar cues in order to drive PGC lineage specification in vitro. ES cells were differentiated as aggregates (embryoid bodies (EBs)), a process that is thought to recapitulate the early stages of embryogenesis by providing an environment conducive to the spontaneous emergence of multiple cell lineages. To date, we have shown that EBs can also support the spontaneous emergence of cells expressing PGC markers. Expression analysis was performed on EBs from 1 to 30 days in culture. PGC markers, including nanog, dazl, fragilis, stella and SSEA1, are expressed in undifferentiated ES cells, but rapidly become undetectable in EBs as the constituent ES cells undergo differentiation. The spontaneous emergence of cells expressing these markers occurred only following long-term EB culture. This indicates a lag in the signalling normally required for PGC specification. In vivo, the lack of BMP4, its receptor (ALK-2) or downstream signalling molecules (Smad 1 and 5), results in the absence of PGCs in embryos. Therefore, in order to enhance PGC specification in our in vitro system, we have added BMP4 into the culture media. Under these conditions, the emergence of cells expressing PGC markers occurs at both an apparently higher efficiency and in a shorter time period. This suggests that BMP4 response pathways are present within the EB context and, when activated, can direct PGC specification. Thus, by recapitulating an in vivo physical and biochemical environment, we are able to direct PGC lineage specification in vitro.

512 IN VITRO PRODUCTION OF HEPATOCYTES FROM HUMAN EMBRYONIC STEM CELLS

2008 ◽  
Vol 48 ◽  
pp. S193-S194
Author(s):  
N. Funakoshi ◽  
S. Gerbal-Chaloin ◽  
M. Daujat-Chavanieu ◽  
F. Navarro ◽  
P. Maurel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
In Vitro Production ◽  
Vitro Production

THE IN VITRO PRODUCTION OF ANDROGENS BY FOLLICULAR TISSUE OF RABBITS

1964 ◽  
Vol 47 (2) ◽  
pp. 306-313 ◽  
Author(s):  
Denis Gospodarowicz
Keyword(s):  
In Vitro Production ◽  
Vitro Production

ABSTRACT Incubation in vitro of rabbit follicles in separate experiments with dehydroepiandrosterone-14C (DHEA-14C), progesterone-14C and pregnenolone-3H in the presence of FSH gave the following results: 39 % of the radioactivity of DHEA-14C is converted to androstenedione and testosterone, while only 3 % of the radioactivity of either progesterone-14C or pregnenolone-3H is found in the androgen fraction. From the ratio of testosterone to androstenedione formed from the three precursors, the results are interpreted to mean that DHEA and pregnenolone, and not progesterone, are precursors of androgens in the follicle.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

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