293 EMBRYONIC STEM-LIKE CELLS DERIVED FROM PORCINE INNER CELL MASS CELLS ISOLATED BY DIFFERENT METHODS

2008 ◽  
Vol 20 (1) ◽  
pp. 226
Author(s):  
X. A. Wolf ◽  
M. A. Rasmussen ◽  
K. Schauser ◽  
P. Maddox-Hyttel
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryonic Fibroblasts ◽  
Serum Replacement ◽  
Sox2 Expression ◽  
Differentiated Cells

The aim was to examine isolation methods and culture conditions for the establishment of embryonic stem-like cells derived from the inner cell mass (ICM) of porcine embryos. A total of 83 zona pellucida-enclosed (ZPE) and 88 hatched (ZPH) porcine in vivo Days 5–7 blastocysts were assigned for ICM isolation by: (A) manual dissection by needles (ZPE: n = 10; ZPH, n = 15), (B) immunosurgical isolation (ZPE: n = 30; ZPH: n = 10), (C) immunosurgery and manual cleaning (ZPE: n = 11; ZPH: n = 40), and (D) culture of whole blastocysts (ZPE: n = 42; ZPH: n = 23). Culture was done on mouse embryonic fibroblasts (MEF) at 5% O2 (for A–C) and 20% O2 (for D) in DMEM with fetal calf serum (FCS), serum replacement, and leukemia inhibitory factor (LIF). Outgrowth colonies (OC) were evaluated by phase contrast and subjected to either (1) physical passage and RT-PCR for Oct-4, Nanog, and Sox2; or (2) immunocytochemical localization of Oct-4 at Days 6–7. Five OC categories were defined: (I) epiblast-like colonies (multilayered ICM-like with homogeneous nuclear Oct-4 staining), (II) ES-like colonies with few surrounding cells (ES-like cells with homogeneous nuclear Oct-4 staining surrounded by few cells), (III) clearly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4 staining and clear demarcation to many differentiated cells), (IV) poorly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4-staining and poor demarcation to many differentiated cells), and (V) differentiated colonies (heterogeneous cell populations lacking nuclear Oct-4 staining). Oct-4 staining was supported by expression of Oct-4, often associated with Nanog and Sox2. The attachment rates were similar for methods A, B, and C, being higher for ZPH (75–80%) than for ZPE (40–50%) blastocysts. Method D gave 42% attachment for ZPE, but only 23% for ZPH blastocysts at 5% O2, whereas the figures at 20% O2 were 63% and 80%, respectively. Methods B and C gave the highest proportion of OCs in categories II–IV (ES-like cell-containing), and the ZPE-derived OCs exhibited a more homogeneous nuclear Oct-4 staining than the ZPH-derived. Method D gave the highest proportion of category II colonies. Passage was performed from OC categories II–IV (with ES-like cells). For method C, 24 OCs resulted in 18 (75%) passage 1 (P1) colonies. In 11, 6, 3, and 2 cases, they were passed to P2, P3, P4, and P5, respectively, maintaining ES-like morphology before they went into quiescence or differentiation. However, most colonies attained cytoplasmic Oct-4 staining and lost Oct-4, Nanog, and Sox2 expression at P1 or P2; only a single colony maintained Nanog and Sox2 expression up to P3. For isolation method D, 10 OCs resulted in 3 (30%) P1 colonies, but only one continued to P2 before differentiation. In conclusion, all methods consistently gave ES-like OCs. Whole blastocysts at 20% O2 gave the highest attachment rates. However, immunosurgery, eventually followed by manual cleaning, tended to result in the highest proportion of OCs presenting ES-like cells, with the ZPE-derived OCs exhibiting the most homogeneous Oct-4 staining. When subjected to passage, a few colonies maintained ES-like morphology up to P5, but expression of pluripotency markers was lost during the initial passages.

Derivation of human embryonic stem cell lines, towards clinical quality

2006 ◽  
Vol 18 (8) ◽  
pp. 823 ◽  
Author(s):  
Outi Hovatta
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Quality Criteria ◽  
Good Manufacturing Practice ◽  
Clinical Quality ◽  
Serum Replacement ◽  
Hes Cells

Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

2005 ◽  
Vol 17 (2) ◽  
pp. 125 ◽  
Author(s):  
Wilfried A. Kues ◽  
Joseph W. Carnwath ◽  
Heiner Niemann
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Embryos ◽  
Somatic Stem Cells ◽  
Primary Fibroblast ◽  
Cell Therapies

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

185 THE PORCINE EPIBLAST AND NOT THE INNER CELL MASS HAS DEVELOPED CONVENTIONAL PATHWAYS FOR REGULATION OF PLURIPOTENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 191
Author(s):  
V. J. Hall ◽  
J. Christensen ◽  
P. Maddox-Hyttel
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positive Control ◽  
Total Rna ◽  
Weak Expression ◽  
Whole Transcriptome

Pluripotency in mice and human embryonic stem cells is regulated by a number of transcription factors, notably including Oct-4, Sox-2, and Nanog. However, in the pig, previous research indicates that Oct-4 protein and mRNA is not specifically localized to the inner cell mass (ICM) of the zona-intact (ZI) blastocyst. Levels of expression of Nanog mRNA, on the other hand, appear to be low in the ZI blastocyst, and protein has not been detected. Similarly, Sox-2 expression in the ZI blastocyst is relatively low and not specific to the ICM. In this study, we investigated the mRNA expression of Oct-4, Sox-2, and Nanog in D6/D7-derived ZI porcine in vivo-derived blastocysts compared with epiblasts mechanically isolated from hatched D10/D11 in vivo-derived blastocysts. We then investigated components involved in pathways important for regulating pluripotency, including JAK/STAT (i.e. gp130, LIFr), FGF (i.e. bFGF, FGFr1, FGFr2), and BMP (bmp4, smad4) signaling pathways and their downstream targets, stat3, c-myc, c-fos, by using RT-PCR. Sows were artificially inseminated, and embryos were flushed from uteri following slaughter. Single D6/D7 blastocysts (n = 3), single mechanically isolated D10/D11 epiblasts (n = 3), endometrium, and oviduct total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Total RNA from the blastocysts and epiblasts was then amplified to form cDNA using the QuantiTect Whole Transcriptome kit (Qiagen). Positive control tissues (oviduct and endometrium) were reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Primers were designed to span introns in highly homologous sequences to human mRNA. Primers were tested in both oviduct and endometrium tissue, and products were sequenced to confirm specificity. PCR was performed at 55°C for 35 cycles. Results indicate that D6/D7 blastocysts only expressed Oct-4 and not Nanog and Sox-2. In contrast, all 3 transcripts were expressed in D10/D11 epiblasts. The D10/D11 epiblasts also expressed LIFr, bFGF, FGFr1, FGFr2, bmp4, smad4, stat3, c-myc, and c-fos. The cytokine receptor gp130 was only weakly expressed in a single epiblast. In contrast, the earlier stage D6/D7 blastocysts failed to express these messengers with the exception of weak expression of gp130 in all 3 blastocysts, and only a single blastocyst expressed LIFr, smad4, c-myc, and c-fos. In conclusion, this study indicates that the ICM of the porcine D6/D7 ZI blastocyst has not developed pluripotency signaling as observed in mice and humans at this developmental stage. Furthermore, without expression of gp130, the JAK/STAT pathway is unlikely to play a role in regulating pluripotency in the epiblast. It is likely that the later stage epiblast may be more amenable for the derivation of porcine embryonic stem cells.

scholarly journals Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts

Biology Open ◽  
2021 ◽  
Author(s):  
Yao Xiao ◽  
Froylan Sosa ◽  
Pablo J. Ross ◽  
Kenneth E. Diffenderfer ◽  
Peter J. Hansen
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Activin A ◽  
Blastocyst Development ◽  
Sox2 Expression ◽  
Nanog Expression ◽  
Canonical Wnt

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of inner cell mass and epiblast during embryonic development.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

54 Effect of Different Cryopreservation Protocols for Sheep Embryonic Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 166
Author(s):  
N. Ibraimova ◽  
A. Seisenbayeva ◽  
Y. Toishibekov
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Slow Freezing

Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P > 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

2010 ◽  
Vol 191 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Daniel Mesnard ◽  
Daniel B. Constam
Keyword(s):  
Cell Surface ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Axis Formation ◽  
Fluorescent Biosensor ◽  
Membrane Bound ◽  
Biosensor Cell

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

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