Laser Direct-Write of Embryonic Stem Cells and Cells Encapsulated in Alginate Beads for Engineered Biological Constructs

2012 ◽  
Vol 1418 ◽  
Author(s):  
T.B. Phamduy ◽  
A.D. Dias ◽  
N. Abdul Raof ◽  
N.R. Schiele ◽  
D.T. Corr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Alginate Beads ◽  
Stem Cell Marker ◽  
Direct Write ◽  
Pluripotency Marker ◽  
Single Bead ◽  
Substrate Surfaces ◽  
Laser Direct Write

AbstractThe ability to control the deposition of mouse embryonic stem cells (mESCs), and mESCs encapsulated in 200-μm diameter alginate microbeads, into customized patterns has recently been achieved using laser direct-write (LDW). Gelatin-based LDW was utilized to target and reproducibly deposit groups of cells directly onto receiving substrate surfaces. Live/dead staining for cell viability and immunocytochemistry for the pluripotency marker, Oct-4, indicated that transferred mESCs were viable following transfer, and maintained an important embryonic stem cell marker, respectively. LDW was further used to print mESCs encapsulated in hydrogel microbeads into customized patterns on a single-bead basis. Recent efforts have also achieved patterns of discrete co-cultures of mESCs and breast cancer cells in separate hydrogel microbeads. Altogether, we demonstrated the feasibility of LDW to print patterns of mESCs and mESC-microbeads for the biomimetic assembly of engineered cellular constructs and tissue models.

Precise Patterning of Mouse Embryonic Stem Cells on Glass Cover Slips for High-Throughput Analysis Using Laser Direct-Write

2011 ◽  
Author(s):  
Andrew D. Dias ◽  
Nathan R. Schiele ◽  
Brendan M. Carr ◽  
Nurazhani Abdul Raof ◽  
Yubing Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Embryonic Stem ◽  
Direct Write ◽  
Glass Cover ◽  
Computer Aided ◽  
Petri Dishes ◽  
Laser Direct Write

Engineering a microenvironment where the growth substrate and distance between cells are controlled is highly desirable to understand how cellular interactions affect stem cell differentiation. Laser direct-write (LDW) allows rapid and precise placement of living cells via computer-aided design/computer-aided manufacturing (CAD/CAM) control. Application of this technique to study the effects of various stem cell microenvironments on differentiation requires a high-throughput experimental setup [1]. Recently, our lab has developed a gelatin-based LDW method for the precise patterning of sensitive cell types, such as mouse embryonic stem cells (mESCs), at a resolution of about 5 μm [2]. Although viable mESCs were successfully printed with maintained pluripotency, this technique required cells to be patterned onto polystyrene Petri dishes [2,3], which may limit high-throughput efficiency. Moreover, the use of polystyrene Petri dishes requires large quantities of culture medium and is not convenient for biological analysis of mESC differentiation. Therefore, the objective of this study was to adapt the LDW method, without altering its prior success, to transfer patterns of viable mESCs to glass cover slips. However, this adaptation to cover slips could not be achieved through simple downscaling due to the unique challenges of providing sufficient moisture for viable cell transfer while maintaining pattern registry on a cover slip. Once cells have been laser patterned, cover slips can then be moved to a 24-well plate so that separate sets of laser patterned cells can be analyzed in parallel for higher experimental throughput utilizing fewer resources to maintain the cells.

scholarly journals The maintenance of pluripotency following laser direct-write of mouse embryonic stem cells

Biomaterials ◽  
2011 ◽  
Vol 32 (7) ◽  
pp. 1802-1808 ◽  
Author(s):  
Nurazhani Abdul Raof ◽  
Nathan R. Schiele ◽  
Yubing Xie ◽  
Douglas B. Chrisey ◽  
David T. Corr
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Direct Write ◽  
Laser Direct Write

scholarly journals Cops5 safeguards genomic stability of embryonic stem cells through regulating cellular metabolism and DNA repair

2020 ◽  
Vol 117 (5) ◽  
pp. 2519-2525 ◽  
Author(s):  
Peng Li ◽  
Lulu Gao ◽  
Tongxi Cui ◽  
Weiyu Zhang ◽  
Zixin Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Genomic Stability ◽  
Cellular Metabolism ◽  
M Cell ◽  
Pluripotency Marker ◽  
Damage Repair ◽  
Pluripotency Maintenance

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

Cryobiology ◽  
2006 ◽  
Vol 53 (2) ◽  
pp. 194-205 ◽  
Author(s):  
Igor I. Katkov ◽  
Min S. Kim ◽  
Ruchi Bajpai ◽  
Yoav S. Altman ◽  
Marc Mercola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Slow Cooling ◽  
Pluripotency Marker

scholarly journals Gender-specific characteristics of hypertrophic response in cardiomyocytes derived from human embryonic stem cells

2021 ◽  
Vol 13 (2) ◽  
pp. 146-155
Author(s):  
Shiva Ahmadvand ◽  
Ali Osia ◽  
Anna Meyfour ◽  
Sara Pahlavan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Hypertrophic Response ◽  
Pluripotency Marker ◽  
Expression Of Genes ◽  
Y Chromosomes ◽  
Gender Specific

Introduction: Gender-specific phenotypes of the heart were reported with respect to both physiology and pathology. While most differences were associated with the sex hormones, differential expression of genes received special attention, particularly X-Y chromosomes’ genes. Methods: Here, we compared cardiogenesis by gene expression analysis of lineage specific markers and X-Y chromosomes’ genes, during in vitro differentiation of XY and XX human embryonic stem cells (hESC), in a hormone-free setup. Results: Downregulation of pluripotency marker (NANOG) and upregulation of cardiac mesoderm and progenitor markers (GATA4, TBX5, NKX2.5, ISL1) was remained temporally similar in differentiating XY and XX hESCs. Isoproterenol treatment of XY and XX hESC-derived cardiomyocytes (hESCCM) induced hypertrophy in a sex-specific manner, with female cardiomyocytes showing response at higher isoproterenol concentration and a later time point of differentiation. Interestingly, KDM5C as an X-linked gene, was markedly upregulated in both hypertrophied male and female cardiomyocytes. Conclusion: Collectively, our results indicated a temporally identical cardiogenesis, but more susceptibility of XY hESC-CM to hypertrophic stimulus in a hormone-free condition.

213 WNT inhibition by dickkopf WNT signalling pathway inhibitor 1 (DKK1) cannot replace IWR-1 during derivation of bovine embryonic stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 234
Author(s):  
Y. Xiao ◽  
T. Amaral ◽  
P. Tribulo ◽  
K. Diffenderfer ◽  
P. Ross ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Wnt Signalling ◽  
Signalling Pathway ◽  
Bovine Embryos ◽  
Pluripotency Marker ◽  
Wnt Signalling Pathway ◽  
Epiblast Stem Cells ◽  
Esc Derivation

Understanding the signalling pathways involved with derivation of embryonic stem cells could enhance our understanding of pluripotency in pre-implantation embryos. Recently, the small molecule IWR-1 has been shown to promote derivation of mouse epiblast stem cells and pluripotent bovine and porcine embryonic stem cells (ESC). IWR-1 blocks WNT signalling mediated by β-catenin-targeted gene expression through stabilisation of Axin2, a member of the destruction complex that induces β-catenin degradation. Here, we evaluated whether dickkopf WNT signalling pathway inhibitor 1 (DKK1) can replace IWR-1 for establishment of bovine pluripotent ESC. If so, it is likely that the actions of IWR-1 to promote pluripotency involve inhibition of WNT signalling. Treatment of bovine embryos with 100ngmL−1 recombinant human DKK1 beginning at Day 5 of development decreased (P=0.02) immunofluorescent labelling of β-catenin in the resulting blastocysts (n=41-45/group), indicating that bovine embryos are responsive to DKK1 treatment. For ESC derivation, blastocysts were plated on top of feeder cells and cultured in ESC medium supplemented with 2.5 µM IWR-1 (n=21), 100ngmL−1 DKK1 (n=34), or vehicle (n=23). Cells were passaged every 5 to 7 days in their respective treatment medium. Seven days after plating, 57.9±14.7% of blastocysts in IWR-1 ESC medium developed outgrowth, which was lower (P=0.02) than the proportion of blastocysts with outgrowth in DKK1 medium (92.4±5.2%) or vehicle (81.9±10.0%). Outgrowth size did not differ among treatments. Labelling with CDX2 indicated that the majority of cells in outgrowths were trophectoderm cells. Thus, IWR-1 inhibits competence of blastocysts to form trophectoderm outgrowths during derivation of ESC. The percent of blastocysts from which cell lines were derived after 4 passages were 48% (10/21) for IWR-1, 41% (14/34) for DKK1, and 48% (11/23) for vehicle. Immunolabelling for the pluripotency marker SOX2 showed that only cells grown in IWR-1 medium were positive, whereas most of the cells derived in the other two media were not. Thus, IWR-1 could not be replaced by DKK1 for maintaining pluripotency. Immunoreactive β-catenin was abundantly distributed on the membrane of cells cultured with IWR-1 but not with DKK1 or vehicle-treated cells. Thus, β-catenin distribution to the cell membrane is linked with bovine pluripotency. Overall, results indicate that maintenance of pluripotency by IWR-1 may involve mechanisms other than WNT inhibition, and may be related to the localization of β-catenin to the plasma membrane.

scholarly journals ZFP982 confers mouse embryonic stem cell characteristics by regulating expression of Nanog, Zfp42 and Dppa3

2020 ◽  
Author(s):  
Fariba Dehghanian ◽  
Patrick Piero Bovio ◽  
Zohreh Hojati ◽  
Tanja Vogel
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Stem Cell Marker ◽  
Marker Genes ◽  
List Type

AbstractWe here used multi-omics analyses to identify and characterize zinc finger protein 982 (Zfp982) that confers stemness characteristics by regulating expression of Nanog, Zfp42 and Dppa3 in mouse embryonic stem cells (mESC). Network-based expression analyses comparing the transcriptional profiles of mESC and differentiated cells revealed high expression of Zfp982 in stem cells. Moreover, Zfp982 showed transcriptional overlap with Yap1, the major co-activator of the Hippo pathway. Quantitative proteomics and co-immunoprecipitation revealed interaction of ZFP982 with YAP1. ZFP982 used a GCAGAGKC motif to bind to chromatin, for example near the stemness conferring genes Nanog, Zfp42 and Dppa3 as shown by ChIP-seq. Loss-of-function experiments in mESC established that expression of Zfp982 is necessary to maintain stem cell characteristics. Zfp982 expression decreased with progressive differentiation, and knockdown of Zfp982 resulted in neural differentiation of mESC. ZFP982 localized to the nucleus in mESC and translocated to the cytoplasm upon neuronal differentiation. Similarly, YAP1 localized to the cytoplasm upon differentiation, but in mESC YAP1 was present in the nucleus and cytoplasm.Graphical AbstractZFP982 is a regulator of stemness of mouse embryonic stem cells and acts as transcription factor by activating expression of stem cell genes including Nanog, Dppa3 and Zfp42.HighlightsZfp982 is a new mouse stem cell defining marker gene.Zfp982 is co-expressed with Yap1 and stem cell marker genes in mESC.ZFP982 binds to DNA and induces expression of master genes of stemness in mESC.Expression of Zfp982 gene prevents neural differentiation and maintains stem cell characteristics.ZFP982 and YAP1 interact in mESC and translocate to the cytoplasm upon neural differentiation.

scholarly journals Soluble Amyloid Precursor Protein Induces Rapid Neural Differentiation of Human Embryonic Stem Cells

2011 ◽  
Vol 286 (27) ◽  
pp. 24264-24274 ◽  
Author(s):  
Kristine K. Freude ◽  
Mahmud Penjwini ◽  
Joy L. Davis ◽  
Frank M. LaFerla ◽  
Mathew Blurton-Jones
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Amyloid Precursor Protein ◽  
Human Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Precursor Protein ◽  
Stem Cell Marker ◽  
Loss Of Function ◽  
Neural Lineage

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis, we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably, we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media, up to 80% of cells expressed the neural stem cell marker nestin, and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation, we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together, our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein.

Using Laser Direct-Write to Influence Embryoid Body Size on Uniform Substrates

2012 ◽  
Author(s):  
Andrew D. Dias ◽  
Yubing Xie ◽  
Douglas B. Chrisey ◽  
David T. Corr
Keyword(s):  
Stem Cells ◽  
Body Size ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Direct Write ◽  
Cell Type ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Cell Sources ◽  
Laser Direct Write

Embryonic stem cells (ESCs) are capable of self-renewal and have the potential to differentiate into any specialized cell type. Understanding and directing this differentiation potential is critical to producing cell sources for replacement therapies.

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