Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2339-2348 ◽  
Author(s):  
B. Pain ◽  
M.E. Clark ◽  
M. Shen ◽  
H. Nakazawa ◽  
M. Sakurai ◽  
...  
Keyword(s):  
Culture System ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Telomerase Activity ◽  
Specific Antibodies ◽  
Typical Morphology ◽  
Germinal Tissue

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

scholarly journals Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1253-1263 ◽  
Author(s):  
Masanori Hirashima ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
Norihisa Matsuyoshi ◽  
Shin-Ichi Nishikawa
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Vascular Endothelial ◽  
Cell Cell

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

Abstract 1964: Bone Morphogenetic Protein Antagonist “Protein Related to Dan and Cerberus” Promotes Differentiation of Embryonic Stem Cells to Atrial Cardiomyocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jingbo Yan ◽  
Jianyong Hu ◽  
Iris I Mueller ◽  
William H Heaton ◽  
Wan-Der Wang ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bmp Signaling ◽  
Cardiac Differentiation ◽  
Cardiovascular Cells

The molecular factors that regulate cardiac differentiation have been extensively studied, yet, relatively little is known about how cardiomyocytes acquire atrial versus ventricular characteristics. Embryonic stem (ES) cells, which have the potential to differentiate to a wide array of distinct cell types, including most types of cardiovascular cells, offer a pertinent in vitro model to work out the molecular mechanisms of atrial specification and differentiation. We discovered that the secreted antagonist of BMP signaling, Protein Related to Dan and Cerberus (PRDC, also called Gremlin2) leads to a surge in cardiomyocytic differentiation when applied to mouse ES-derived cardiac progenitor cells. This property is unique to PRDC among tested BMP antagonists. Lineage expansion is restricted to cardiomyocytes, with the differentiation of endodermal, blood, endothelial and neuronal cells being unaffected. Using molecular and electrophysiological analyses, we show that PRDC-induced cardiomyocytes acquire atrial characteristics. Consistent with the in vitro results, we found that injection of PRDC mRNA into the developing zebrafish embryo leads to supernumerary contracting areas. The ectopic cardiomyocytes express atrial-, but not ventricular- specific cardiac genes. We determined that PRDC treatment induces the expression of COUP-TFII, a known transcriptional regulator of atrial differentiation, but suppresses Notch signaling. Inhibition of Notch is sufficient to induce atrial-specific genes; however, blocking Notch does not expand the cardiogenic fields. Taken together, our data suggest that antagonism of BMP and Notch signaling by PRDC is a critical early step in the specification, expansion and differentiation of atrial progenitor cells. This information might be relevant for treating atrial degeneration, as well as for understanding the etiology of atrial fibrillation.

scholarly journals An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Li Zhang ◽  
Yenan Wu ◽  
Xiang Li ◽  
Shao Wei ◽  
Yiming Xing ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Neural Cells ◽  
Human Leukemia ◽  
Stem Cell Markers ◽  
Novel Model ◽  
Chicken Embryonic Stem Cells

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro. Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro.

scholarly journals Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

2000 ◽  
Vol 20 (20) ◽  
pp. 7419-7426 ◽  
Author(s):  
Sara R. Cherry ◽  
D. Biniszkiewicz ◽  
L. van Parijs ◽  
D. Baltimore ◽  
R. Jaenisch
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Transcriptional Repression ◽  
Fluorescent Protein ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Embryonic Stem Cells Differentiated in Vitro as a Novel Source of Cells for Transplantation

1996 ◽  
Vol 5 (2) ◽  
pp. 131-143 ◽  
Author(s):  
Jonathan Dinsmore ◽  
Judson Ratliff ◽  
Terry Deacon ◽  
Peyman Pakzaba ◽  
Douglas Jacoby ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Es Cells ◽  
Embryonic Stem ◽  
Muscle Cells ◽  
Cell Types ◽  
Cell Type ◽  
Skeletal Myoblasts ◽  
Muscle Cell Type

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of γ-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntington's disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.

Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 335-342 ◽  
Author(s):  
Fei Li ◽  
Shijiang Lu ◽  
Loyda Vida ◽  
James A. Thomson ◽  
George R. Honig
Keyword(s):  
Rhesus Monkey ◽  
Bone Morphogenetic Protein ◽  
Culture System ◽  
Es Cells ◽  
Regulatory Protein ◽  
Embryonic Stem ◽  
Bone Morphogenetic Protein 4 ◽  
Hematopoietic Differentiation ◽  
Morphogenetic Protein

A cell culture system consisting of mouse S17 stromal cells supplemented with cytokines was developed for hematopoietic differentiation of rhesus monkey embryonic stem (ES) cells. The differentiated colonies that formed contained clusters of hematopoietic-like cells, as well as structures similar in appearance to embryonic blood islands. When this culture system was supplemented with bone morphogenetic protein 4 (BMP-4), the numbers of primary hematopoietic clusters increased by an average of 15 fold. The primary hematopoietic clusters containing clonogenic precursors (expandable hematopoietic clusters) increased by 18 fold. Immunofluorescence analysis showed that a substantial percentage of the hematopoietic-like cells were CD34+, with morphologic features of undifferentiated blast cells. Enrichment of the CD34+ cells was associated with enhanced stromal-dependent, cytokine-driven formation of cobblestone colonies on secondary plating. The hematopoietic identity of the precursors was further indicated by their expression of genes associated with hematopoietic differentiation, as well as morphologic assessments that showed erythroid and myeloid lineages among the progeny cells. In addition, reverse transcriptase–polymerase chain reaction analysis of BMP-4–treated rhesus monkey ES cells demonstrated an up-regulation of early-expressed genes responsible for embryonic hematopoiesis and angiogenesis during the first 7 days of culture. These observations suggest that embryonic mesoderm regulatory protein may mimic physiologic signals that are required for the onset of embryonic hematopoiesis and stem cell formation in rhesus monkey ES cells.

scholarly journals Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture

Reproduction ◽  
2006 ◽  
Vol 132 (2) ◽  
pp. 257-263 ◽  
Author(s):  
T Amano ◽  
M Gertsenstein ◽  
A Nagy ◽  
H Kurihara ◽  
H Suzuki
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Potential ◽  
Developmental Capacity ◽  
Developmental Rates

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.

scholarly journals Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Khodadad Khodadadi ◽  
Huseyin Sumer ◽  
Maryam Pashaiasl ◽  
Susan Lim ◽  
Mark Williamson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histochemical Staining ◽  
Chromosome Count ◽  
Embryoid Bodies ◽  
Retroviral Transduction

Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report ofin vivodifferentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors usingOCT4, SOX2, andKLF4in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression ofOCT4, SOX2 NANOG, andSTAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers bothin vitroby embryoid bodies (EBs) formation andin vivoby teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.

224 DERIVATION AND IMMUNOLOGICAL CHARACTERISTICS OF PORCINE EMBRYONIC STEM-LIKE CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
J.-E. Kim ◽  
H.-Y. Son ◽  
H.-S. Kim ◽  
E.-G. Lee ◽  
C.-K. Lee
Keyword(s):  
Stem Cell ◽  
Cell Surface ◽  
Immune Responses ◽  
Es Cells ◽  
Embryonic Stem ◽  
Swine Leukocyte Antigen ◽  
Leukocyte Antigen ◽  
Typical Morphology

Pluripotent cells from porcine blastocysts can be used to serve as an in vitro model as well as an unlimited cell source for therapeutic xenotransplantation. In xenotransplantation, foreign organs and tissues are rejected because of the immune responses in the recipient. This rejection is determined by the naturally occurring cell surface antigen, major histocompatibility antigen (MHC), knowledge of which is essential to advance our understanding of the processes implicated in immune responses. In mouse and human embryonic stem (ES) cells, MHC expression patterns are changed upon their differentiation in vitro. Therefore, immunological characteristics of undifferentiated and/or differentiated pluripotent ES-like cells derived from porcine blastocysts could provide valuable information to xenotransplantation research. For such purposes, swine leukocyte antigen (SLA) isotype and its expression pattern were determined in porcine ES-like cells. Porcine inner cell masses from Day 7 in vivo-produced blastocysts were isolated by immunosurgery and seeded on a feeder layer. Isolated cells were cultured in a mixture of DMEM : HF-10 (1 : 1) with 15% FBS, 1.7 mM l-glutamine, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1000 U mL-1 leukemia inhibitory factor, 40 ng mL-1 stem cell factor, and 20 ng mL-1 basic fibroblast growth factor in humidified environment of 5% CO2 in air at 38�C. Porcine ES-like cells showing typical morphology were subpassaged by physical separation. Embryoid body (EB) formation was induced by culturing small clumps of cells by the hanging drop method. Total RNA was extracted from porcine ES-like cell and EBs. PCR amplification was performed by using locus-specific primer pairs for swine leukocyte antigen (SLA) 1 and 2, followed by sequencing of amplified DNA fragments. These sequences were compared with known SLA sequences, and their SLA allele types were determined. Porcine ES-like cells showed the typical morphology of ES cells, closer to that of human than mouse cells. They showed typical expression of pluripotent stem cell markers, determined by immunochemistry and PCR analysis. When porcine ES-like cells formed EBs, differentiation specific markers of all 3 germ layers, �%-fetoprotein (endoderm marker), neurofilament (ectoderm marker), and cardiac troponin1 (mesoderm), were detected in the EBs. In porcine ES-like cells, two new alleles (SLA-1*06ck01 and *06ck02, and SLA-2*06ck01 and *06ck02) were identified. Therefore, our porcine ES-like cell line showed heterozygous allele isotypes in SLA-1 and SLA-2. EBs derived from porcine ES-like cells showed the same sequencing results, indicating no alteration of SLA-1 and SLA-2 isotypes in porcine ES-like cells and their differentiated derivatives. Further experiments for determining SLA-3 allele isotype in porcine ES-like cells and EBs and cell surface expression of the SLA antigens in these cells will be performed. In conclusion, porcine ES-like cells were established from in vivo-derived blastocysts and their SLA isotypes were consistent regardless of their differentiation.

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