104. PROLIFERATION ABILITY, TELOMERASE ACTIVITY AND MOLECULAR CHARACTERIZATION OF PLURIPOTENT CELL LINES FROM IVF AND PARTHENOGENTIC PIG EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 23
Author(s):  
T. A. L. Brevini ◽  
G. Pennarossa ◽  
L. Attanasio ◽  
B. Gasparrini ◽  
F. Gandolfi
Keyword(s):  
Cell Lines ◽  
Doubling Time ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Pluripotency Markers ◽  
Proliferation Ability ◽  
Receptor Beta

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.

275 DERIVATION OF PLURIPOTENT CELL LINES FROM PIG EMBRYOS: IN VITRO-FERTILIZED V. PARTHENOGENTIC ACTIVATION

2009 ◽  
Vol 21 (1) ◽  
pp. 235
Author(s):  
F. Gandolfi ◽  
G. Pennarossa ◽  
L. Attanasio ◽  
S. Antonini ◽  
B. Gasparrini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Telomerase Activity ◽  
Animal Biotechnology ◽  
Pluripotency Markers ◽  
Biology Research ◽  
Parthenogenetic Embryos

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217). Table 1. Supported by: Prin 2005, 2006.

287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 223 ◽  
Author(s):  
T. Lonergan ◽  
A. Harvey ◽  
J. Zhao ◽  
B. Bavister ◽  
C. Brenner
Keyword(s):  
Cell Lines ◽  
Complex I ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Fibroblast ◽  
Es Cell ◽  
Cell Content ◽  
Stem Cell Lines

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Chiaki Sano ◽  
Asako Matsumoto ◽  
Eimei Sato ◽  
Emiko Fukui ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Long Term Culture ◽  
Oct4 Expression ◽  
Feeder Layers

SummaryEmbryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

228 ESTABLISHMENT OF EMBRYONIC STEM-LIKE CELL LINES IN RABBITS

2007 ◽  
Vol 19 (1) ◽  
pp. 230 ◽  
Author(s):  
Y.-W. Ou ◽  
K.-H. Lee ◽  
L.-R. Chen ◽  
P.-C. Tang ◽  
H.-F. Guu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Immunocytochemical Staining ◽  
Es Cell ◽  
Feeder Layers ◽  
Feeder Group

Embryonic stem (ES) cells are pluripotent cells from the inner cell mass (ICM) of the blastocyst. They are capable of differentiating to various cell types, such as neural cells, cardiocytes, hepatic cells, and germ cells. The aim of this study was to establish rabbit ES cell lines as an animal model for human diseases. Blastocysts were collected from New Zealand White rabbits during Days 4 to 5 after breeding. After removal of the mucin coat and the zona pellucida by pronase, the embryos were directly cultured in ES cell medium on mitomycin C-treated mouse embryonic fibroblast (MEF) or STO feeder layers. In Experiment 1, the efficiencies of 2 different feeder layers, MEF and STO, in generating rabbit ES cell lines were compared. Six blastocysts were used for each STO and MEF feeder group. The primary ICM colonies were formed in 67% (4/6) of the cultures on the STO and 83% (5/6) on the MEF. Sixty percent of those primary colonies (3/5) were successfully grown into ES-like cell lines in the MEF feeder group. However, no cell lines were established on the STO feeder. In Experiment 2, whole blastocysts or ICMs isolated by immunosurgery were cultured to establish ES cell lines. A total of 21 blastocysts were recovered from 2 does. Eighteen whole blastocysts and 3 isolated ICMs were cultured on the MEF feeders. Twelve (67%) of the cultured whole blastocysts formed primary ICM colonies, of which 5 (42%) of the cultures continuously propagated and formed ES-like cell lines. In the immunosurgical group, 2 of the 3 isolated ICMs formed primary colonies but only 1 ES-like cell line was established. A total of 9 ES-like cell lines maintained morphological undifferentiation after 14 passages and expressed alkaline phosphatase activity. Seven of the 9 ES-like cells expressed Oct-4 and the stage-specific embryonic antigen-4 (SSEA-4) as detected by immunocytochemical staining. Two cell lines were further induced to differentiate into embryoid bodies in suspension culture. Another 3 cell lines were injected into SCID mice and one of them formed a teratoma. The competence of generating chimeric rabbits and the teratogenicity of the established ES-like cell lines are under evaluation. In conclusion, rabbit ES-like cells were efficiently generated and whole-blastocyst culturing on the MEF feeder appeared to be a preferred method for the isolation and maintenance of rabbit ES-like cell lines.

scholarly journals Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Tielens ◽  
B Verhasselt ◽  
J Liu ◽  
M Dhont ◽  
J Van Der Elst ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Stem Cell Lines

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Sincein vitroculture may influence Oct-4 expression, we investigated to what extent blastocysts culturedin vitrofrom the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We comparedin vivowithin vitroderived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells’ differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of thein vitroderived blastocysts, 17% gave rise to ES cells vs 38% of thein vivoblastocysts. Six-day old outgrowths fromin vivodeveloped blastocysts expressed Oct-4 in 55% of the cases vs 31% of thein vitroderived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collectedin vivoblastocysts compared toin vitrocultured blastocysts.In vitrocultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level thanin vivoblastocysts.

scholarly journals Pluripotency and differentiation of embryonic stem cells

2020 ◽  
Vol 185 ◽  
pp. 04034
Author(s):  
Yinyin Liu ◽  
Haibo Zhao ◽  
Liang Liang ◽  
Peilei Fan ◽  
Yujia Zhao ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regenerative Medicine ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Self Renewal ◽  
Extracellular Signals ◽  
And Function

Mouse embryonic stem (ES) cells derive from the inner cell mass of an early embryo called blastocyst, making them promising resource for regenerative medicine. They possess two unique properties: self-renewal and pluripotency. Different ways can be used to assess which extracellular signal and factor inside ES cells has an impact on the pluripotency of ES cells. Nowadays, many extracellular signals and transcription factors have been identified, such as extracellular signals like LIF and transcription factors like Oct4. Studying the mechanism and function of these factors offers great insight and advance our understanding of pluripotency and self-renewal and thus shed light on regenerative medicine.

302 IDENTIFICATION OF 3i TARGET MOLECULES AND THEIR INVOLVEMENT IN PORCINE PLURIPOTENCY NETWORKS

2013 ◽  
Vol 25 (1) ◽  
pp. 298
Author(s):  
G. Pennarossa ◽  
S. Maffei ◽  
M. M. Rahman ◽  
F. Gandolfi ◽  
T. A. L. Brevini
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Large Animal ◽  
Rt Pcr ◽  
Pluripotent Cell ◽  
Pluripotency Markers ◽  
Target Molecules

Recent studies have shown that the use of specific inhibitors for signalling pathways known to drive murine embryonic stem cell (ESC) differentiation may represent a tool to derive and maintain pluripotent cell lines. The application of this novel approach could provide a new strategy to overcome the limitations still existing for the derivation of ESC in large animal species. These molecules, also known as 3i factors, include CHIR99021 (GSK3 inhibitor), PD173074 (FGF inhibitor) and PD0325901 (MAPK/ERK kinase or MEK inhibitor). However only scattered information are available on the involvement of these pathways in the maintenance of pluripotency in domestic animals. The aim of this study was to investigate the presence of receptors for these inhibitors in porcine inner cell mass (ICM) and to isolate and culture porcine pluripotent lines in a serum-free medium supplemented with the 3i factors, without any additional growth factor. Ovaries were collected at the local abattoir and cumulus–oocyte complexes (COC) were aspirated from antral follicles. In vitro maturation was then performed for 46 h. Frozen–thawed spermatozoa were purified and live spermatozoa were co-cultured with denuded oocytes for 24 h. Putative embryos were cultured in NCSU-23 medium until the blastocyst stage and then subjected to immuno-surgery. Isolated ICM were analysed by RT-PCR. Poly(A)+RNA was extracted using Dynabeads® mRNA DIRECT Micro-kit (Invitrogen, Carlsbad, CA, USA) and immediately reverse-transcribed with Superscript™ II Reverse Transcriptase (Invitrogen). Specific primers were designed for FGF4, FGFR-1, FGFR-2, FGFR-4, GSK3, and MEK genes. The PCR amplified products were sequenced and aligned using ClustalW. The RT-PCR results showed that porcine ICM actively transcribe for GSK3, MEK, FGFR-2, FGFR-4, and FGF4 genes, whereas no signal was detectable for FGFR-1. Based on these observations, IVF-derived ICM were plated onto inactivated STO feeder cells and cultured in N2B27 medium supplemented with 3 µM CHIR99021, 100 nM PD173074, and 0.4 µM PD0325901. Outgrowth formation was monitored and cells were passaged to a new STO monolayer every 7 days, as previously described (Brevini et al. 2010 Stem Cell Rev.). Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 15 passages. The results obtained indicate that porcine cells cultured in 3i medium, without additional growth factors, expressed pluripotency markers; namely OCT4, NANOG, SOX2, and REX1, preserving their pluripotent state over time. Our data indicate that porcine ICM express 3i factor target molecules. In agreement with this, the use of 3i medium allows the establishment and proliferation of pluripotent cell lines. Together, these findings suggest the involvement of the GSK3, FGF, and MEK pathways in porcine pluripotency network and advocate the use of the 3i medium as an efficient tool for ESC derivation in porcine. Supported by NetLiPS Project ID: 30190629.

197 PRODUCTION OF IN VITRO- AND IN VIVO-DERIVED CAT BLASTOCYSTS FOR THE ESTABLISHMENT OF FELINE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 207 ◽  
Author(s):  
J. Kehler ◽  
M. Roelke-Parker ◽  
B. Pukazhenthi ◽  
W. Swanson ◽  
C. Ware ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Feeder Layers

Identification and characterization of spontaneously occurring genetic diseases in cats has permitted the development of valuable models for testing potential treatments of similar human diseases. With the near completion of the feline genome project, establishment of pluripotential feline embryonic stem (ES) cells would facilitate the targeting of specific genetic loci to produce new feline medical models. Two approaches were used to produce feline blastocysts in an attempt to establish feline ES cells in culture. Naive queens were superovulated with an intramuscular (i.m.) injection of 150 IU of equine chorionic gonadotropin (eCG) followed by an i.m. injection of 100 IU of human chorionic gonadotropin (hCG) 80 h later; follicles were aspirated laparoscopically 24-26 h later for subsequent in vitro fertilization (IVF). On average, 29 mature cumulus oocyte cell complexes (COCs) were recovered from each queen. IVF was performed in 50 microliter drops of complete Hams F-10 medium containing 30 000 fresh, motile sperm. COCs were cultured overnight in 5% carbon dioxide at 38�C, and residual adherent cumulus cells were removed 12 to 16 h later by trituration in 0.1% hyaluronidase. Embryos were cultured in fresh drops of Hams F-10, and on average 25% developed to the early blastocyst stage after 7 days. Alternatively, estrus was induced in queens with a single i.m. injection of 100 IU of eCG, and then 72 h later queens were permitted six supervised matings with a fertile tom over the next two days. Queens underwent ovariohysterectomy 7 days after their first copulation, and compacted morulae and early blastocysts were flushed from the oviducts and uterine horns. On average, eight embryos were recovered from the reproductive tract of each queen. Both in vivo- and in vitro-matured blastocysts were subsequently cultured in standard mouse ES cell medium on inactivated mouse embryonic fibroblasts. When they failed to hatch in culture after 3 days, a 0.5% pronase solution was used to dissolve the zonae pellucidae under microscopic visualization. Denuded expanded blastocysts adhered to the heterotypic feeder layer and primary inner cell mass (ICM) outgrowths formed within 4 days. Outgrowths were mechanically disaggregated into small clusters of 15 to 20 cells and re-plated on fresh feeders. These colonies grew slowly and were transferred after one week onto new feeder layers. The addition of murine or human recombinant leukemia inhibitory factor had no effect on the survival and proliferation of primary outgrowths or subsequent colonies. After 3 weeks, all colonies derived from both in vivo- and in vitro-matured blastocysts had either differentiated or died. Additional experiments are ongoing to test the effects of homotypic feeder layers and alternative growth factors on promoting the establishment and survival of feline ES cell lines. Ultimately, germline transmission of any putative feline ES cell lines will need to be demonstrated in vivo for their utility in gene targeting experiments to be realized.

297 DERIVATION AND CHARACTERIZATION OF THE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER-DERIVED BOVINE EMBRYONIC STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 246
Author(s):  
S. H. Jeong ◽  
H. S. Kim ◽  
H. Lee ◽  
K. J. Uh ◽  
S. H. Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Mass Culture ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Trophoblast Cells

Bovine transgenic embryonic stem (ES) cells have not been reported yet because it seems that the derivation methods and the culture conditions for the inner cell mass are neither consistent nor optimized. Isolation of inner cell mass and primary culture of ES colonies is a critical step toward the establishment of authentic bovine ES cell lines. Herein, we reconstructed somatic cell nuclear transferred (SCNT) bovine blastocysts carrying a vector expressing the human INF-α gene, and isolated inner cell masses to derive transgenic bovine embryonic stem cells. In addition, we added 2 inhibitors, inhibition (2i system) of the mitogen-activated protein kinase (Erk1/2) cascade, PD0325901(3 Î1/4M), and of glycogen synthase kinase 3, CHIR99021 (1 Î1/4M), in the inner cell mass primary culture to check reliability of the 2i system for bovine ES culture. The 2 inhibitors made the morphology of colonies more intact, and primary colonies were better maintained in early passages. However, there were no significant effects on the attachment rate and maintenance in late passages (percent of percent over 3 passages: 2i system, 21/38 (55.3%); control, 22/42 (33.3%); P < 0.05). Inner cell masses were isolated mechanically and subcultured by an enzymatic in primary inner cell mass culture. Massive growth of trophoblast cells appears to inhibit inner cell mass growth, so hatching and hatched blastocysts were cut with a needle to remove trophoblast cells. Poor quality blastocysts were attached by the whole seeding method, and the margin trophoblast cells were consecutively removed in early passages. Established bovine ES cells express alkaline phosphatase, Oct-4, SSEA1, SSEA4, Tra-1–60, and Tra-1–81. We confirmed pluripotent gene expression of bovine ES like cells; Oct-4, SSEA1, and Rex 1 were positive, but trophoblast marker CDX2 was negative. This study shows that the 2i system is a reasonable method for use during inner cell mass culture in early passages. We established 6 transgenic nuclear transfer bovine ES cell lines with the 2i system and 4 in vitro fertilized bovine ES cell lines (all were over 10 passages).

277 ESTABLISHMENT OF EMBRYONIC STEM CELL LINES DERIVED FROM A EGFP-TRANSGENIC MOUSE AND THEIR SURVIVAL IN THE COCHLEA OF C57BL/6 MICE

2008 ◽  
Vol 20 (1) ◽  
pp. 218
Author(s):  
K. S. Ahn ◽  
S. J. Jeon ◽  
J. Y. Jung ◽  
T. Choi ◽  
S. J. Choi ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Transgenic Mouse ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Nerve Fibers ◽  
Embryoid Bodies ◽  
Ganglion Neurons

Embryonic stem (ES) cells isolated from inner cell mass cells of blastocyst-stage embryos are capable of differentiating into various cell lineages. Transplantation of these cells may potentially be a treatment for many degenerative diseases. Such cell therapy has often been tested using allografts of ES cells in mice. However, it has been difficult to locate transplanted ES cells and to avoid the rejection of allogeneic ES cells by the host. The aims of this study were to establish ES cell lines ubiquitously expressing enhanced green fluorescent protein (EGFP) and to test survival of ES cells in allografts into the cochlea of inbred C57BL/6 mice. Nine hatched blastocysts collected from a C57BL/6-green mouse that ubiquitously expresses transgene EGFP were plated onto an inactivated STO feeder layer. Two putative ES-like colonies were obtained from the plated blastocysts, and repeated subculture of these colonies produced two cell lines expressing EGFP. The cell lines possessed typical characteristics of ES cells, including densely packed colonies of the cells with prominent nucleoli, a high nuclear-cytoplasmic ratio, and high alkaline phosphatase activity. In suspension culture, these cells formed simple and cystic embryoid bodies. Undifferentiated EGFP-transgenic ES cells (106 cells per mouse) were injected into the cochlea of five C57BL/6 mice deafened by gentamycin treatment. Although no behavioral changes were noticed until four weeks after the transplantation, histological study revealed that grafted cells survived in the scala media of all injected mice. Incorporation of the cells expressing EGFP into the host was found along the auditory nerve fibers close to the organ of Corti. Such incorporation was also discovered in the area of the spiral ganglion neurons, cochlear sensory epithelia, and stria vascularis. Morphology and size of the cells varied depending on their sites of incorporation. The results from the present study demonstrate that, due to their survival in transplantation without allogeneic rejection as well as ubiquitous and stable expression of EGFP, ES cells from an EGFP-transgenic mouse may be a useful means of studying cell therapy.

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