In vitro differentiation of embryonic stem cells into glial cells and functional neurons

1995 ◽  
Vol 108 (10) ◽  
pp. 3181-3188 ◽  
Author(s):  
A. Fraichard ◽  
O. Chassande ◽  
G. Bilbaut ◽  
C. Dehay ◽  
P. Savatier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Glial Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Acid Decarboxylase ◽  
Isolated Cells ◽  
Specific Antigens

Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyte- or oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltage-dependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.

scholarly journals Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Kimia Hosseini ◽  
Emilia Lekholm ◽  
Aikeremu Ahemaiti ◽  
Robert Fredriksson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Glial Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Neuronal Cultures ◽  
Short Period

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
S. Wang ◽  
X. Tang ◽  
Y. Niu ◽  
H. Chen ◽  
T. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Speed ◽  
Es Cells ◽  
Research Work ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

Study of the C60 Fullerene on Differentiation of Mouse Embryonic Stem Cells

2012 ◽  
Vol 529-530 ◽  
pp. 385-390
Author(s):  
Koichi Imai ◽  
Fumio Watari ◽  
Kazuaki Nakamura ◽  
Akito Tanoue
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Experimental Method ◽  
Es Cells ◽  
Embryonic Stem ◽  
Future Generations ◽  
C60 Fullerene ◽  
Biological Safety ◽  
Mouse Es Cells

The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We have conducted anin vitroembryotoxicity test with mouse ES cells to examine the embryotoxicities of various nanomaterials. In this study, the C60 fullerene did not influence the differentiation of ES-D3 cells and "non embryotoxicity". In the future, the biological safety should be comprehensively examined by improving dispersion in medium.

scholarly journals Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Angiogenesis ◽  
2021 ◽  
Author(s):  
Anna Gualandris ◽  
Alessio Noghero ◽  
Davide Cora’ ◽  
Elena Astanina ◽  
Marco Arese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture Medium ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endothelial Differentiation ◽  
Loss Of Function ◽  
Pure Cultures

AbstractEmbryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

231 HISTOCOMPATIBLE PARTHENOGENETIC EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 232
Author(s):  
A. Yabuuchi ◽  
K. Kitai ◽  
A. Takeuchi ◽  
P. Lerou ◽  
K. Ng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Full Complement ◽  
Teratoma Formation ◽  
High Level ◽  
Selection Of

Organ or tissue transplantation is the preferred treatment for numerous diseases but is hindered by immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are possible sources of histocompatible cells and tissues. We have developed two ways of isolating pES cells that carry the full complement of major histocompatibility complex (MHC) antigens of the oocyte donors. One method entails activation of oocytes after blockade of karyokinesis in meiosis II, followed by selection of predominantly homozygous pES cells that have undergone recombination in their MHC antigen region to restore the heterozygous maternal MHC genotype (parthenote recombinant, or prES cells). The second method involves activation of immature oocytes after blockade of karyokinesis of meiosis I, followed by selection of predominantly heterozygous pES lines that retain the MHC genotype of the oocyte donor (parthenote clone recombinant, or pcrES cells). The cells are pluripotent by several criteria: teratoma formation, in vitro differentiation into hematopoietic elements, and high-level skin chimerism in blastocyst chimeras. Breeding of 8 founder females and examination of over 700 progeny failed to demonstrate germ line transmission of the pES cells. Injection of over 50 tetraploid embryos with these lines and embryo transfer have failed to support full gestational development. However, differentiated tissues from these pluripotent ES cells engraft when transplanted into genetically matched immunocompetent recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.

The in vitro production and characterization of neutrophils from embryonic stem cells

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 852-859 ◽  
Author(s):  
Jonathan G. Lieber ◽  
Saiphone Webb ◽  
Benjamin T. Suratt ◽  
Scott K. Young ◽  
Gary L. Johnson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Specific Antigen ◽  
Embryonic Stem ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Functional Evaluation ◽  
In Vitro Production

AbstractAn embryonic stem (ES) cell/OP9 coculture system for the effective production of functional neutrophils is described. A 3-step differentiation strategy was developed that uses liquid culture, enabling reliable and abundant production of neutrophils at high purity without the need of sorting for isolation of mature neutrophils. Use of the OP9 stromal cell line significantly enhances the number, percentage, and duration of differentiated neutrophils produced from embryonic stem cells. Effective and sustained differentiation of ES cells into neutrophils provides a useful model system for studying neutrophil differentiation and function and the factors that regulate them. Morphologic and functional evaluation of these ES-derived neutrophils indicates that large numbers of mature neutrophils can be produced from pluripotent ES cells in vitro. Specifically, their morphology, ability to produce superoxides, flux calcium, undergo chemotaxis in response to macrophage inflammatory protein 2 (MIP-2), stain for the granulocyte-specific marker–specific chloroacetate esterase, and contain the neutrophil-specific markers Gr-1 and the mouse neutrophil-specific antigen indicates that they are comparable with purified mouse bone marrow neutrophils. They also express gelatinase and lactoferrin granule proteins. During the differentiation of these ES-derived neutrophils, regional areas of neutrophil production can be identified that have been designated as neutrophil generating regions (NGRs).

Enrichment of blood from embryonic stem cells in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 563 ◽  
Author(s):  
Andrew C. Perkins
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Leukaemia Inhibitory Factor ◽  
Chimeric Mouse ◽  
Differentiation Potential

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.

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