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

McKee, Christina; Brown, Christina; Chaudhry, G. Rasul

doi:10.3390/cells8121650

Cited by 11 publications

(23 citation statements)

References 90 publications

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“…Induction of YAP expression was shown to aid in the long-term survival and expansion of human ESCs in 2-D culture [71]. Furthermore, we have shown induction of naïve-like pluripotency in primed human ESCs grown in 3-D culture, which was abolished upon inhibition of YAP [37]. These observations further suggest a mechanistic role of cytoskeleton remodeling and mechanical signaling in the maintenance of pluripotency.…”

Section: Discussionsupporting

confidence: 59%

“…However, upregulation of select core pluripotent markers has been observed in 3-D culture of mouse [36,54] as well as human ESCs [55,56], suggesting a dependence on scaffold composition and culture conditions. We have reported upregulation of select naïve pluripotent markers in primed [37] and naïve human ESCs In both mouse and human ESCs, focal adhesion and integrin signaling serve pleiotropic roles in anchorage, contractility, survival, proliferation as well as support or inhibition of self-renewal [63]. Our study revealed upregulation in 3-D grown cells of several genes such as FLNC, COL4, CAV1, and SERPINE1, as well as ITGB1, ILK, and AKT1, which have been reported to be associated with actin cytoskeleton remodeling [64], and integrin mediated focal adhesion activation [65], respectively.…”

Section: Discussionmentioning

confidence: 96%

“…8-arm PEG polymers functionalized with thiol and acrylate end groups were purchased from JenKem Technology USA (Plano, TX), and stored in the dark at -20°C. PEG-8-SH and PEG-8-Acr polymers were combined to facilitate self-assembly, as previously described [37]. Briefly, PEG-8-SH and PEG-8-Acr were separately dissolved in culture media at 2.5 w/v% polymer concentration and mixed thoroughly at a 1:1 molar ratio in the presence of oxygen for polymerization via a thiol-Michael addition reaction.…”

Section: Self-assembly Of 3-d Scaffolds Via Thiol-michael Addition Rementioning

confidence: 99%

“…Therefore, synthetic biomaterials may prove to be ideal for the development of the defined 3-D culture conditions needed for the propagation of human ESCs. We have previously demonstrated 3-D culture of naïve mouse ESCs [36] and primed human ESCs [37] using hydrogel scaffolds that resulted in modulation of gene expression, and maintenance of pluripotent cells. In this study, we promoting their use in basic and applied applications.…”

Section: Introductionmentioning

confidence: 99%

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

McKee

Brown

Bakshi

et al. 2020

Preprint

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Derivation 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.

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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 96%

Section: Self-assembly Of 3-d Scaffolds Via Thiol-michael Addition Rementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

McKee

Brown

Bakshi

et al. 2020

Preprint

Self Cite

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“…This Special Issue includes articles highlighting the effective use of induced pluripotent stem cells (iPSCs) to develop cardiac microtissue [ 1 ], neurospheres [ 2 ], and cortical progenitors [ 3 ]. In addition, this Special Issue contains work on scaffolds with mesenchymal stem cells (MSCs) [ 4 ] and embryonic stem cells (ESCs) [ 5 ]. Furthermore, research on generating intestinal organoids from stem cells [ 6 , 7 ] and nephrogenesis studies utilizing ESCs [ 8 ] are included in this Special Issue, along with a comprehensive review on the 3D culture of hematopoietic stem cells (HSCs) [ 9 ].…”

mentioning

confidence: 99%

3D Stem Cell Culture

Ylöstalo

2020

Cells

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Much interest has been directed towards stem cells, both in basic and translational research, to understand basic stem cell biology and to develop new therapies for many disorders. In general, stem cells can be cultured with relative ease, however, most common culture methods for stem cells employ 2D techniques using plastic. These cultures do not well represent the stem cell niches in the body, which are delicate microenvironments composed of not only stem cells, but also supporting stromal cells, extracellular matrix, and growth factors. Therefore, researchers and clinicians have been seeking optimal stem cell preparations for basic research and clinical applications, and these might be attainable through 3D culture of stem cells. The 3D cultures recapitulate the in vivo cell-to-cell and cell-to-matrix interactions more effectively, and the cells in 3D cultures exhibit many unique and desirable characteristics. The culture of stem cells in 3D may employ various matrices or scaffolds, in addition to the cells, to support the complex structures. The goal of this Special Issue is to bring together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cells and culture techniques, and also highlight stem cell preparations for possible novel therapeutic applications.

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Universal Peptide Hydrogel for Scalable Physiological Formation and Bioprinting of 3D Spheroids from Human Induced Pluripotent Stem Cells

Liu

et al. 2021

Adv Funct Materials

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Human induced pluripotent stem cells (hiPSCs) are used for drug discoveries, disease modeling and show great potential for human organ regeneration. 3D culture methods have been demonstrated to be an advanced approach compared to the traditional monolayer (2D) method. Here, a self-healing universal peptide hydrogel is reported for manufacturing physiologically formed hiPSC spheroids. With 100 000 hiPSCs encapsulated in 500 µL hydrogel, ≈50 000 spheroids mL −1 (diameter 20-50 µm) are generated in 5 d. The spheroids in the universal peptide hydrogel are viable (85-96%) and show superior pluripotency and differentiation potential based on multiple biomarkers. Cell performance is influenced by the degradability of the hydrogel but not by gel strength. Without postprinting crosslinking aided by UV or visible lights or chemicals, various patterns are easily extruded from a simple star to a kidney-like organ shape using the universal peptide hydrogel bioink showing acceptable printability. A 20.0 × 20.0 × 0.75 mm 3 sheet is finally printed with the universal peptide hydrogel bioink encapsulating hiPSCs and cultured for multiple days, and the hiPSC spheroids are physiologically formed and well maintained.

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

Cited by 11 publications

References 90 publications

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

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

3D Stem Cell Culture

Universal Peptide Hydrogel for Scalable Physiological Formation and Bioprinting of 3D Spheroids from Human Induced Pluripotent Stem Cells

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