scholarly journals Transcriptomic analysis of naïve human embryonic stem cells cultured in three-dimensional PEG scaffolds

2020 ◽  
Author(s):  
Christina McKee ◽  
Christina Brown ◽  
Shreeya Bakshi ◽  
Keegan Walker ◽  
Chhabi K. Govind ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Molecular Characteristics ◽  
Regulation Of Transcription

AbstractDerivation of primed and naïve human embryonic stem cells (ESCs) have prompted an increased interest in devising culture conditions for maintaining their pluripotency and differential potential. Naïve ESCs are characterized by improved viability, proliferation, and differentiation capacity in comparison to primed ESCs. However, traditional two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, which results in altered morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of human ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffolds not only maintained the naïve state, but also supported long-term growth for up to 3 weeks without requiring routine passaging and manipulation. 3-D grown cells exhibited upregulation of core (OCT4, NANOG, and SOX2) and naïve (KLF17, KLF4, TFCP2L1, DPPA3, and DNMT3L) genes. These genes returned to normal levels when 3-D grown cells were propagated under 2-D culture conditions. Examination of RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown Elf1 cells. Gene Ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, as well as β-catenin-TCF complex assembly, extracellular matrix organization, and chromatin remodeling in 3-D grown Elf1 cells. 3-D culture conditions also induced upregulation of genes associated with several signaling pathways including Wnt signaling and focal adhesion. However, p53 signaling pathway associated genes were downregulated under these culture conditions. Our findings provide insight into the possible mechanisms of prolonged self-renewal as well as upregulation of pluripotent genes stimulated by the transduction of mechanical signals from the 3-D microenvironment.

scholarly journals Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 21
Author(s):  
Christina McKee ◽  
Christina Brown ◽  
Shreeya Bakshi ◽  
Keegan Walker ◽  
Chhabi K. Govind ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Molecular Characteristics ◽  
Regulation Of Transcription ◽  
Human Escs

Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon the combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported the long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings, for the first time, provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.

scholarly journals Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1650 ◽  
Author(s):  
Christina McKee ◽  
Christina Brown ◽  
G. Rasul Chaudhry
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Extended Period ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Self Assembling

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.

Expression profiles of miRNAs in human embryonic stem cells during hepatocyte differentiation

2011 ◽  
Vol 41 (2) ◽  
pp. 170-183 ◽  
Author(s):  
Nury Kim ◽  
Hyemin Kim ◽  
Inkyung Jung ◽  
Yeji Kim ◽  
Dongsup Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Hepatocyte Differentiation

In vivo Differentiation of Human Embryonic Stem Cells

2007 ◽  
pp. 121-147
Author(s):  
Scott A. Noggle ◽  
Francesca M. Spagnoli ◽  
Ali H. Brivanlou
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
In Vivo Differentiation

GRGD‐decorated three‐dimensional nanoworm hydrogels for culturing human embryonic stem cells

2019 ◽  
Vol 57 (18) ◽  
pp. 1956-1963 ◽  
Author(s):  
Stefanie Kessel ◽  
Nilay Thakar ◽  
Zhongfan Jia ◽  
Ernst J. Wolvetang ◽  
Michael J. Monteiro
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Three Dimensional ◽  
Embryonic Stem

100. IN VIVO DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO UTERINE TISSUE

2009 ◽  
Vol 21 (9) ◽  
pp. 19
Author(s):  
L. Ye ◽  
R. Mayberry ◽  
E. Stanley ◽  
A. Elefanty ◽  
C. Gargett
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epithelial Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mesenchymal Cells ◽  
Neonatal Mouse ◽  
Endometrial Tissue ◽  
Human Epithelial Cells

The endometrium undergoes cyclic regeneration. This regeneration has been attributed to adult stem progenitor cells and developmental mechanisms [1, 2]. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and on early origins of adult endometrial disease. The lack of human fetal endometrial tissue has impeded research in early human endometrial development. We hypothesized that directed differentiation of human embryonic stem cells (hESC) to human endometrial tissue by neonatal mouse uterine mesenchyme represents a novel system to study early development of human endometrium. Recent studies have shown that the neonatal mouse uterine mesenchyme is extremely inductive and undergoes reciprocal signalling with human endometrial epithelial cells [3]. Our aim is to establish a xenograft tissue recombination protocol based on a model for human prostate tissue differentiation using hESC [4]. Our method involved formation of embryoid body (EB) with GFP labelled hESC (ENVY) [5] for recombination with 2x0.5mm pieces of epithelial-free uterine mesenchyme from postnatal day 1 mice. Upon fusion in culture, the recombinant tissue is grafted under the kidney capsule of NOD/SCID mice for 4-12 weeks and monitored by in-vivo imaging. Immunohistochemical analysis of recombinant grafts 4 weeks post transplantation (n=4) revealed immature CK8+CK18+Hoxa10+ human epithelial cells surrounded by mouse mesenchymal cells suggesting differentiation of hESC to epithelial cells possibly of endometrial lineage. The ER+PR+SMA+Hoxa10+ mouse mesenchymal cells surrounding human glands differentiated into SMA+ cells possibly via reciprocal signalling from human epithelial cells. At 8 weeks, we found several CK18+/Hoxa10+ human glands co-expressing CA125. These glands are supported by Hoxa10+ human stromal cells. Further experiments are underway to induce the expression of ER and PR in Hoxa10+ epithelial cells which will be crucial in revealing their endometrial lineage.

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