scholarly journals Direct synthesis of self-organized blastocyst-like cysts derived from human pluripotent stem cells

2019 ◽  
Author(s):  
Xiaopeng Wen ◽  
Shiho Terada ◽  
Koki Yoshimoto ◽  
Ken-ichiro Kamei
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Direct Synthesis ◽  
Cell Mass ◽  
Human Pluripotent Stem Cells ◽  
Molecular Signatures ◽  
Poly Ethylene Glycol ◽  
Self Organized ◽  
Poly Ethylene

AbstractWe introduce a simple, robust and scalable method to generate self-organized blastocyst-like cysts (soBLCs) from human pluripotent stem cells (hPSCs). We use a copolymer hydrogel of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG). hPSC aggregates with a diameter of approximately 117.2 ± 5.1 µm are cultured in a medium supplemented with a hydrogel and a serum for three days. Molecular signatures in the medium revealed the generation of trophoblasts and inner cell mass at specific positions in the soBLCs.

scholarly journals An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

2017 ◽  
Author(s):  
Jianying Guo ◽  
Dacheng Ma ◽  
Rujin Huang ◽  
Jia Ming ◽  
Min Ye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Genetic Manipulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Human Pluripotent Stem Cells ◽  
Hesc Line ◽  
Transcription Activator

AbstractHuman pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
B. P. Telugu ◽  
T. Ezashi ◽  
A. Alexenko ◽  
S. Lee ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Induced Pluripotent

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

Exploring early differentiation and pluripotency in domestic animals

2017 ◽  
Vol 29 (1) ◽  
pp. 101 ◽  
Author(s):  
R. Michael Roberts ◽  
Ye Yuan ◽  
Toshihiko Ezashi
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Short Review ◽  
Potential Usefulness ◽  
Meat Production ◽  
Human Escs ◽  
Domestic Species

This short review describes some general features of the origins of the pluripotent inner cell mass and epiblast during the early development of eutherian mammals and the two kinds of embryonic stem cell (ESC), naïve and primed type, that have been produced from these structures. We point out that the derivation of pluripotent stem cells from domesticated species continues to be fraught with difficulties, most likely because the culture requirements of these cells are distinct from those of mouse and human ESCs. Generation of induced pluripotent stem cells (iPSCs) from the domesticated species has been more straightforward, although the majority of the iPSC lines remain dependent on the continued expression of one or more integrated reprogramming genes. Although hope for the potential usefulness of these cells in genetic modification of livestock and other domestic species has dimmed, ESCs and iPSCs remain our best source of self-renewing populations of pluripotent cells, with potential usefulness in preserving and propagating valuable animal breeds and making contributions to fields such as regenerative medicine, toxicology and even laboratory meat production.

scholarly journals Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

2016 ◽  
Vol 6 (4) ◽  
pp. 437-446 ◽  
Author(s):  
Ge Guo ◽  
Ferdinand von Meyenn ◽  
Fatima Santos ◽  
Yaoyao Chen ◽  
Wolf Reik ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Isolated Cells

305 CONSTRUCTION OF TRANSCRIPTION FACTOR GENES FOR REPROGRAMMING OF ADULT GOAT FIBROBLAST CELLS FOR PRODUCTION OF INDUCED PLURIPOTENT STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 249
Author(s):  
D. Kumar ◽  
D. Malakar ◽  
R. Dutta ◽  
S. Garg ◽  
S. Sahu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Embryonic stem cells (ESC) are derived from the inner cell mass of blastocysts and proliferate extensively while maintaining pluripotency. They can be used for the treatment of juvenile diabetes, Parkinson’s disease, heart failure, and spinal cord injury. However, the use of embryos and tissue rejection remain concerns for ESC transplantation. Reprogramming of somatic cells may be done by different methods such as somatic cell nuclear transfer (Wilmut et al. 1997), fusion of somatic cells (Cowen et al. 2005), treatment with the extract of the pluripotent stem cells (Johnson Rajasingh 2008), and by the stable ectopic expression of defined factors in the somatic cells (Takahashi and Yamanaka 2006). Several transcription factors, including Oct3/4 (Nichols et al. 1998; Niwa et al. 2000), Sox2 (Avilion et al. 2003), and Nanog (Chambers et al. 2003; Mitsui et al. 2003), function in the maintenance of pluripotency in both early embryos and ESC. Takahashi and Yamanaka reported reprogramming the fibroblast cells into stem cells by introducing Oct3/4, Sox2, c-Myc, and Klf4 in mouse embryonic and adult fibroblasts. Yu et al. (2007) demonstrated that four transcription factors (OCT-4, SOX2, NANOG, and LIN28) are sufficient to reprogramme human somatic cells to pluripotent stem cells that exhibit the essential characteristics of ESC. Nakagawa et al. (2008) used three factors (OCT3/4, SOX2, and KLF4) for human iPS cell production from somatic cells. We are trying to reprogramme the adult goat fibroblast cells in induced pluripotent stem cells by using ectopic expression of transcription factors such as Oct-4, Sox2, Nanog, and Lin28. We collected the ovaries from a slaughtered animal from Delhi and collected the oocytes from ovaries. Then after the collection, A and B grade oocytes were selected. Selected oocytes were processed and incubated in in vitro maturation media for 24 h. We collected semen from a male goat, and it was processed and capacitated in sperm TALP. Capacitated sperms were used for IVF of the in vitro matured oocytes in ferTALP. After 12 h sperm were washed from oocytes in embryo developing media (EDM), and oocytes were cultured (in vitro) in EDM. After 24 h cleavage occurred. The cleaved embryos were cultured for 6 to 7 days. At the 7th day, we got blastocysts. From these blastocysts, inner cell mass was isolated enzymatically and cultured to get ESC. The ESC were cultured for 7 passages and used for RNA isolation. The RNA was isolated from these stem cells by the Trizol method. Complementary DNA was prepared by RT-PCR. Using gene-specific primer for Oct-4, Nanog, and Sox2, DNA was amplified. The DNA for the Oct-4, Nanog, and Sox2 genes was cloned in pJET cloning vector and transformed in Top10 E. coli competence cells. After screening, plasmid was isolated and sent for sequencing. Sequences were analysed and the complete open reading frame was created for Oct-4, Nanog, and Sox2.

scholarly journals Leukemia Inhibitory Factor (LIF)-dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos

2011 ◽  
Vol 286 (33) ◽  
pp. 28948-28953 ◽  
Author(s):  
Bhanu Prakash V. L. Telugu ◽  
Toshihiko Ezashi ◽  
Sunilima Sinha ◽  
Andrei P. Alexenko ◽  
Lee Spate ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Inhibitory Factor

scholarly journals Epigenetic resetting of human pluripotency

2017 ◽  
Author(s):  
Ge Guo ◽  
Ferdinand von Meyenn ◽  
Maria Rostovskaya ◽  
James Clarke ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Doubling Time ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Linked Gene ◽  
Histone Deacetylase Inhibition ◽  
Lineage Differentiation

SUMMARYMuch attention has focussed on conversion of human pluripotent stem cells (PSC) to a more naive developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 hours. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from primed PSC and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to the level in the ICM but is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of bi-allelic X-linked gene transcription indicates re-activation of the silenced X chromosome. On re-conversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

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