204 SPONTANEOUS DIFFERENTIATION OF PORCINE INNER CELL MASS-DERIVED CELLS INTO CELLS DISPLAYING NEURAL AND GLIAL MARKERS

2006 ◽  
Vol 18 (2) ◽  
pp. 210 ◽  
Author(s):  
K. Schauser ◽  
S. Friis ◽  
M. Schmidt ◽  
P. Maddox-Hyttel
Keyword(s):  
Progenitor Cells ◽  
Glial Cells ◽  
Neural Differentiation ◽  
Neural Progenitor Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Es Cell ◽  
Cytoplasmic Staining

The pig is needed as a model for human embryonic stem (ES) cell therapy. Our aims were to isolate, culture, and characterize porcine inner cell mass (ICM) derived cells. Porcine blastocysts (n = 22) were flushed from sows on Day 8 after insemination and two blastocysts were immediately fixed. A total of 18 (90%) ICMs were microsurgically isolated from the remaining blastocysts and cultured on mitomycin-inactivated mouse embryonic fibroblasts (SLN cells) in a human ES cell medium without (n = 9) or with 106 U/mL leukemia inhibitory factor (LIF, n = 9). The colonies were inspected by stereomicroscopy every second day. After approximately one week, ES-like portions of the primary outgrowth colonies were passaged physically. Subsequently, passages were performed approximately once a week. Colonies from different passages were fixed in 4% paraformaldehyde and processed for immunocytochemistry together with the blastocysts. LIF had no apparent effect on rates of attachment and growth during initial outgrowth and after passages, or on differentiation patterns. A total of 15 (83%) ICMs attached during the outgrowth culture and 12 (80%) of these developed ES-like portions, i.e. compact masses of small tightly packed cells. In blastocysts, nuclei of the ICM stained exclusively for Oct-4. After 6 days of culture, however, Oct-4 staining was lacking in outgrowth colonies. After 14 days of culture, 6 out of 13 passage (P) 1 colonies had developed morphological characteristics compatible with neural differentiation, i.e. large bipolar perikarya and long axon-like structures; after one month, 15 out of 17 P2-4 colonies displayed neural differentiation. Cells in such colonies displayed cytoplasmic staining for �-III-tubulin in the perikarya and axon-like extensions. After 2-3 months of culture, cell populations in colonies with neural differentation displayed cytoplasmic staining for the intermediate filaments nestin (marker for neural progenitor cells) and vimentin (in the nervous system a marker for glial cells), and cytoplasmic staining for �-III-tubulin and TUC-4 (markers of post-mitotic neurons). Double-immunostaining revealed a co-localization pattern suggesting the existence of a heterogeneous neural cell population that included neural progenitor cells (staining for nestin only), maturing neural progenitor cells (staining for nestin throughout the cytoplasm combined with �-III tubulin in the axon-like extensions only), early neurons (staining for �-III-tubulin, TUC-4, and nestin in the complete cytoplasm), and glial cells (staining for vimentin alone or in combination with nestin). At no time point studied so far were the neural transcription factors Pax6 and Sox2 detected. In conclusion, ICMs were efficiently isolated from Day 8 porcine blastocysts and attached to feeder cells. However, during initial outgrowth culture they lost their Oct-4 expression, and over the subsequent passages they developed into a heterogeneous population of cells at different stages of neural differentiation.

scholarly journals Phenotype and Stability of Neural Differentiation of Androgenetic Murine ES Cell-Derived Neural Progenitor Cells

Cell Medicine ◽  
2013 ◽  
Vol 5 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Wanja Wolber ◽  
Ruhel Ahmad ◽  
Soon Won Choi ◽  
Sigrid Eckardt ◽  
K. John Mclaughlin ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Neural Differentiation ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Es Cell

Transplantation of Motoneuron-Enriched Neural Cells Derived from Mouse Embryonic Stem Cells Improves Motor Function of Hemiplegic Mice

2003 ◽  
Vol 12 (5) ◽  
pp. 457-468 ◽  
Author(s):  
Shunmei Chiba ◽  
Yasumasa Iwasaki ◽  
Hiroaki Sekino ◽  
Noboru Suzuki
Keyword(s):  
Motor Cortex ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Cortical Region ◽  
Neural Cells ◽  
Es Cell ◽  
Neuroprotective Effects

Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cell-derived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an “island of the mature neuronal cells” of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.

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.

REPROGRAMMING OF PORCINE EPIBLAST-DERIVED NEURAL PROGENITOR CELLS TO PLURIPOTENCY

2012 ◽  
Vol 24 (1) ◽  
pp. 289
Author(s):  
M. A. Rasmussen ◽  
V. J. Hall ◽  
S. G. Petkov ◽  
O. Ujhelly ◽  
M. Pirity ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Autologous Transplantation ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Embryoid Bodies ◽  
Advanced Technology ◽  
Feeder Cells ◽  
Pluripotency Genes

Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK, and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and sub-cultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC, and KLF4, as evaluated by immunocytochemistry, and expression of NANOG, SSEA-1, and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR, and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project “PartnErS” (218205; 204, 523) and EU FP7 Stem Cell Project “PluriSys” (223485).

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.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

Stromal derived factor-1α in hippocampus radial glial cells in vitro regulates the migration of neural progenitor cells

2015 ◽  
Vol 39 (6) ◽  
pp. 750-758 ◽  
Author(s):  
Hui Ding ◽  
Guo-Hua Jin ◽  
Lin-Qing Zou ◽  
Xiao-Qing Zhang ◽  
Hao-Ming Li ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Glial Cells ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Radial Glial Cells ◽  
Radial Glial

scholarly journals Temporal and epigenetic regulation of neurodevelopmental plasticity

2007 ◽  
Vol 363 (1489) ◽  
pp. 23-38 ◽  
Author(s):  
Nicholas D Allen
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Developmental Plasticity ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Neural Progenitors ◽  
Developmental Potential ◽  
Neural Lineage

The anticipated therapeutic uses of neural stem cells depend on their ability to retain a certain level of developmental plasticity. In particular, cells must respond to developmental manipulations designed to specify precise neural fates. Studies in vivo and in vitro have shown that the developmental potential of neural progenitor cells changes and becomes progressively restricted with time. For in vitro cultured neural progenitors, it is those derived from embryonic stem cells that exhibit the greatest developmental potential. It is clear that both extrinsic and intrinsic mechanisms determine the developmental potential of neural progenitors and that epigenetic, or chromatin structural, changes regulate and coordinate hierarchical changes in fate-determining gene expression. Here, we review the temporal changes in developmental plasticity of neural progenitor cells and discuss the epigenetic mechanisms that underpin these changes. We propose that understanding the processes of epigenetic programming within the neural lineage is likely to lead to the development of more rationale strategies for cell reprogramming that may be used to expand the developmental potential of otherwise restricted progenitor populations.

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