Comparative Parallel Multi-Omics Analysis During the Induction of Pluripotent and Trophectoderm States

Following fertilization, totipotent cells divide to generate two compartments in the early embryo: the inner cell mass (ICM) and trophectoderm (TE). It is only at the 32-64 -cell stage when a clear segregation between the two cell-types is observed, suggesting a ‘T’-shaped model of specification. Here, we examine whether the acquisition of these two states in vitro by nuclear reprogramming share similar dynamics/trajectories. We conducted a comparative parallel multi-omics analysis on cells undergoing reprogramming to Induced pluripotent stem cells (iPSCs) and induced trophoblast stem cells (TSCs), and examined their transcriptome, methylome, chromatin accessibility and activity and genomic stability. Our analysis revealed that cells undergoing reprogramming to pluripotency and TSC state exhibit specific trajectories from the onset of the process, suggesting ‘V’-shaped model. Using these analyses, not only we could describe in detail the various trajectories toward the two states, we also identified previously unknown stage-specific reprogramming markers as well as markers for faithful reprogramming and reprogramming blockers. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these specific regions are initially open but then retain inactive by the elimination of the histone mark, H3K27ac.

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

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab305 ◽

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.

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223 GENERATION OF INTERSPECIES CHIMERAS BETWEEN PRIMATE INDUCED PLURIPOTENT STEM CELLS AND PORCINE PARTHENOGENETIC EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab223 ◽

2016 ◽

Vol 28 (2) ◽

pp. 243

Author(s):

M. Nowak-Imialek ◽

S. Wunderlich ◽

D. Herrmann ◽

S. Klein ◽

U. Baulain ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

In Vitro Culture ◽

Inner Cell Mass ◽

Cell Mass ◽

Culture Conditions ◽

Serum Replacement ◽

Induced Pluripotent ◽

Parthenogenetic Embryos

The availability of human induced pluripotent stem cell (hiPSC) paves the way to generate regenerative tissue or organs from patient’s own stem cells. The production of chimeric piglets carrying organs that are entirely derived from human stem cells, or at least have a high contribution of human cells or tissues, could be used as a new tissue or an organ replacement in the future treatment of the patients. Here, we produced porcine-nonhuman primate chimeric embryos to assess the feasibility of the potential use of human iPSC for production of human stem cell-derived organs in pigs. Because in vitro culture conditions for cynomolgus monkey iPSC and porcine blastocysts are different, we first identified an effective in vitro culture system for porcine blastocysts and monkey iPSC. We compared blastocyst rates (Days 7 and 8) and number of cells of porcine parthenogenetic blastocysts (Day 8) cultured in 8 different monkey iPSC media and in porcine zygote medium 3 (PZM-3). The best developmental rates of porcine blastocysts were achieved in Knockout DMEM+20% serum replacement monkey medium (iPS 20% medium; N = 65, n = 3). The number of blastocysts on Day 8 cultured in iPS 20% medium was significantly higher (91%; P < 0.05) than in the commonly used porcine PZM-3 medium (65%). We found significantly fewer (P < 0.05) degenerate porcine embryos on Day 8 after culture in iPS 20% medium (9%) compared to PZM-3 (35%). The number of nuclei per blastocyst in iPS 20% medium (88 nuclei; N = 30, n = 3) was significantly higher (P < 0.0001) than in the PZM-3 medium (57 nuclei; N = 54, n = 3). Therefore, we decided to use iPS 20% medium for culture of porcine blastocysts injected with monkey iPSC. Thereafter, we injected clusters of 10 to 15 monkey iPSC transgenic with AAVS1-CAG-Venus into porcine parthenogenetic embryos from Days 4 and 6. Interspecies chimeras were cultured in iPS 20% medium for 24 (for Day 6 embryos) or 48 h (for Day 4 embryos) and observed by confocal microscopy to determine the proportion of Venus-expressing monkey iPSC in porcine embryos. Approximately 37% of blastocysts contained Venus-positive cells after injection of Day 6 embryos (N = 133, n = 4). In contrast, injection into porcine embryos from Day 4 resulted in 73% of Venus-positive blastocysts (N = 69, n = 3). Finally, we investigated proliferation and survival of monkey iPSC in interspecies chimeras after blastocyst plating onto murine fibroblasts. Chimeric blastocyst outgrowth resulted in Venus-expressing monkey iPSC proliferating over 1 week in culture. Outgrowths of all chimeric blastocysts established distinct but separate monkey and porcine stem cell colonies. Here, we optimized the culture conditions for an in vitro interspecies chimera assay in which monkey iPSC are able to survive in porcine embryos. Integration of monkey iPSC to host inner cell mass is relevant for the further contribution to the embryo development. Therefore, to verify this, analysis of cell-cell connection between monkey iPSC and porcine blastocysts and experiments using vivo-derived embryos are currently underway.

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Generation of Induced Pluripotent Stem (iPS) Cells by Nuclear Reprogramming

Stem Cells International ◽

10.4061/2011/619583 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 10

Author(s):

Dilip Dey ◽

Gregory R. D. Evans

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Adult Stem Cells ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Types ◽

Nuclear Reprogramming ◽

Differentiated Cells ◽

Reprogramming Factors ◽

Induced Pluripotent

During embryonic development pluripotency is progressively lost irreversibly by cell division, differentiation, migration and organ formation. Terminally differentiated cells do not generate other kinds of cells. Pluripotent stem cells are a great source of varying cell types that are used for tissue regeneration or repair of damaged tissue. The pluripotent stem cells can be derived from inner cell mass of blastocyte but its application is limited due to ethical concerns. The recent discovery of iPS with defined reprogramming factors has initiated a flurry of works on stem cell in various laboratories. The pluripotent cells can be derived from various differentiated adult cells as well as from adult stem cells by nuclear reprogramming, somatic cell nuclear transfer etc. In this review article, different aspects of nuclear reprogramming are discussed.

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Pluripotent stem cells: induction and self-renewal

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0213 ◽

2018 ◽

Vol 373 (1750) ◽

pp. 20170213 ◽

Cited By ~ 9

Author(s):

R. Abu-Dawud ◽

N. Graffmann ◽

S. Ferber ◽

W. Wruck ◽

J. Adjaye

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Inner Cell Mass ◽

Meta Analysis ◽

Cell Mass ◽

Somatic Cells ◽

Embryonic Stem ◽

Cell Types ◽

Self Renewal

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers—mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

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Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

Scientific Reports ◽

10.1038/s41598-021-90653-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marino Maemura ◽

Hiroaki Taketsuru ◽

Yuki Nakajima ◽

Ruiqi Shao ◽

Ayaka Kakihara ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Mouse Embryos ◽

Primitive Endoderm ◽

Cell Stage ◽

Supportive Environment ◽

Mouse Zygotes ◽

Single Blastomeres ◽

Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

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Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

International Journal of Molecular Sciences ◽

10.3390/ijms22094334 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4334

Author(s):

Katrina Albert ◽

Jonna Niskanen ◽

Sara Kälvälä ◽

Šárka Lehtonen

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Neurodegenerative Disease ◽

Induced Pluripotent Stem Cells ◽

Glial Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Human Ipscs ◽

Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

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A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽

10.1242/dev.118.4.1353 ◽

1993 ◽

Vol 118 (4) ◽

pp. 1353-1361

Author(s):

J.M. Baltz ◽

J.D. Biggers ◽

C. Lechene

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Types ◽

Preimplantation Embryos ◽

Developmentally Regulated ◽

Cell Stage ◽

K Concentration ◽

Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

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Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽

10.1242/dev.121.3.743 ◽

1995 ◽

Vol 121 (3) ◽

pp. 743-753 ◽

Cited By ~ 9

Author(s):

J.E. Collins ◽

J.E. Lorimer ◽

D.R. Garrod ◽

S.C. Pidsley ◽

R.S. Buxton ◽

...

Keyword(s):

Protein Expression ◽

Molecular Mechanisms ◽

Inner Cell Mass ◽

Splice Variants ◽

Cell Mass ◽

Cumulus Cells ◽

Early Embryo ◽

Preimplantation Embryos ◽

Cell Stage ◽

Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

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Stem cell defects in parthenogenetic peri-implantation embryos

Development ◽

10.1242/dev.121.7.2069 ◽

1995 ◽

Vol 121 (7) ◽

pp. 2069-2077

Author(s):

E.D. Newman-Smith ◽

Z. Werb

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Types ◽

Insulin Like Growth Factor ◽

Embryonic Antigen ◽

Parietal Endoderm

Mouse embryos containing only maternal chromosomes (parthenotes) develop abnormally in vivo, usually failing at the peri-implantation stage. We have analyzed the development of parthenote embryos by using an inner cell mass (ICM) outgrowth assay that mimics peri-implantation development. ICMs from normal embryos maintained undifferentiated stem cells positive for stage-specific embryonic antigen-1 and Rex-1 while differentiating into a variety of cell types, including visceral endoderm-like cells and parietal endoderm cells. In contrast, ICMs from parthenotes failed to maintain undifferentiated stem cells and differentiated almost exclusively into parietal endoderm. This suggests that parthenote ICMs have a defect that leads to differentiation, rather than maintenance, of the stem cells, and a defect that leads to a parietal endoderm fate for the stem cells. To test the hypothesis that the ICM population is not maintained owing to a lack of proliferation of the stem cells, we investigated whether mitogenic agents were able to maintain the ICM population in parthenotes. When parthenote blastocysts were supplied with the insulin-like growth factor-1 receptor (Igf-1r) and insulin-like growth factor-2 (Igf-2), two genes not detectable in parthenote blastocysts by in situ hybridization, the ICM population was maintained. Similarly, culture of parthenote blastocysts in medium conditioned by embryonic fibroblasts and supplemented with the maternal factor leukemia inhibitory factor maintained the ICM population. However, once this growth factor-rich medium was removed, the parthenote ICM cells still differentiated predominantly into parietal endoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

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Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽

10.1242/dev.29.3.601 ◽

1973 ◽

Vol 29 (3) ◽

pp. 601-615

Author(s):

M. H. L. Snow

Keyword(s):

Specific Activity ◽

Inner Cell Mass ◽

Cell Damage ◽

Cell Mass ◽

Cell Number ◽

Mouse Embryos ◽

Abnormal Development ◽

Tritium Concentration ◽

Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P < 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

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