Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells

Joshi, Ramila; Buchanan, James C.; Paruchuri, Sailaja; Morris, Nathan; Tavana, Hossein

doi:10.1371/journal.pone.0166316

Cited by 12 publications

(15 citation statements)

References 66 publications

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“…28,37 Persistent and increasing gene expression of these factors in differentiating mESCs is consistent with their roles in development of specific neural cells that takes place during the second week of co-culture of mESCs and PA6 cells. 38,39 In summary, the fold change data in Fig 5a–b suggests that a group of 12 factors self-regulate neural cell differentiation of mESCs in this co-culture system.…”

Section: Resultsmentioning

confidence: 73%

Self-regulatory factors of embryonic stem cells in co-culture with stromal cells enhance neural differentiation

2017

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Embryonic stem cells (ESCs), due to their intrinsic capability to generate somatic cells of all three germ layers, are the potential sources of neural cells for cell replacement therapies. However, the empirical differentiation protocols and the lack of mechanistic understanding of the neural differentiation of ESCs has limited the utility of ESCs as a developmental model or as a cell source for neural cell population for replacement therapies. Coculturing ESCs with stromal cells is one of the extensively used methods to induce neural differentiation. Despite several studies attempting to identify neural inducing factors in stromal cell induced neural differentiation, self-regulatory effects of ESCs in the neural differentiation process remains unexplored. For the first time, we elucidate the self-regulatory role of mESCs on their neural cell differentiation by supplementing conditioned media from differentiating mESCs to the mESCs-PA6 cocultures and quantitatively evaluating the change in neural differentiation. Moreover, we use statistical tools to analyze the expressions of various growth and trophic factors and distinguish the factors produced primarily by PA6 cells versus mESCs in coculture. We observed that addition of the medium containing mESCs-secreted factors to the single mESCs colony cocultured with PA6 cells significantly enhanced the neural differentiation of mESCs compared to the medium extracted from the stromal cells only. Hierarchical clustering of gene expression data from PA6 and cocultured mESCs segregated two group of factors that are produced by the stromal cells and differentiating mESCs. Identifying the major soluble factors that drive and regulate the neural differentiation process in mESCs-PA6 coculture niche will help understand the molecular mechanisms of neural development. Moreover, it can be the first step toward developing novel protocols to differentiate stem cells with mESCs derived factors supplementation without using feeder cells with greater efficiency compared to existing approaches.

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Section: Resultsmentioning

confidence: 73%

Self-regulatory factors of embryonic stem cells in co-culture with stromal cells enhance neural differentiation

2017

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Section: Applications: Toward Functional Artificial Llpsmentioning

confidence: 99%

Cell‐Inspired All‐Aqueous Microfluidics: From Intracellular Liquid–Liquid Phase Separation toward Advanced Biomaterials

et al. 2020

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Living cells have evolved over billions of years to develop structural and functional complexity with numerous intracellular compartments that are formed due to liquid–liquid phase separation (LLPS). Discovery of the amazing and vital roles of cells in life has sparked tremendous efforts to investigate and replicate the intracellular LLPS. Among them, all‐aqueous emulsions are a minimalistic liquid model that recapitulates the structural and functional features of membraneless organelles and protocells. Here, an emerging all‐aqueous microfluidic technology derived from micrometer‐scaled manipulation of LLPS is presented; the technology enables the state‐of‐art design of advanced biomaterials with exquisite structural proficiency and diversified biological functions. Moreover, a variety of emerging biomedical applications, including encapsulation and delivery of bioactive gradients, fabrication of artificial membraneless organelles, as well as printing and assembly of predesigned cell patterns and living tissues, are inspired by their cellular counterparts. Finally, the challenges and perspectives for further advancing the cell‐inspired all‐aqueous microfluidics toward a more powerful and versatile platform are discussed, particularly regarding new opportunities in multidisciplinary fundamental research and biomedical applications.

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“…Proliferation of pluripotent mESCs and resulting multipotent neural stem and progenitor cells causes growth and formation of the colonies during the first week of culture. Cells subsequently acquire less proliferative neuronal or glial progenitor states or terminally differentiate to neurons, astrocytes, or oligodendrocytes during the second week (Joshi, Buchanan, Paruchuri, Morris, & Tavana, ; Kawasaki et al, ). Our temporal gene expression study showed transition in the state of mESCs and supported this explanation (Figure S1–S3).…”

Section: Resultsmentioning

confidence: 99%

“…Our previous study showed that expression of specific markers of neural stem and progenitor cells, such as Nestin and TuJ, and neurite processes is detectable in mESCs colonies starting from the fourth day of coculture with PA6 cells (Joshi et al, ). The expression of neural markers gradually increased until Day 8 and plateaued thereafter.…”

Section: Resultsmentioning

confidence: 99%

Stem cell colony interspacing effect on differentiation to neural cells

Joshi

Fuller

Mosadegh

et al. 2018

J Tissue Eng Regen Med

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Efforts to enhance the efficiency of neural differentiation of stem cells are primarily focused on exogenous modulation of physical niche parameters such as surface topography and extracellular matrix proteins, or addition of certain growth factors or small molecules to culture media. We report a novel neurogenic niche to enhance the neural differentiation of embryonic stem cells (ESCs) without any external intervention by micropatterning ESCs into spatially organized colonies of controlled size and interspacing. Using an aqueous two-phase system cell microprinting technology, we generated pairs of uniformly sized isolated ESC colonies at defined interspacing distances over a layer of differentiation-inducing stromal cells. Our comprehensive analysis of temporal expression of neural genes and proteins of cells in colony pairs showed that interspacing two colonies at approximately 0.66 times the colony diameter (0.66D) significantly enhanced neural differentiation of ESCs. Cells in these colonies displayed higher expression of neural genes and proteins and formed thick neurite bundles between the two colonies. A computational model of spatial distribution of soluble factors of cells in interspaced colony pairs showed that the enhanced neural differentiation is due to the presence of stable concentration gradients of soluble signalling factors between the two colonies. Our results indicate that culturing ESCs in colony pairs with defined interspacing is a promising approach to efficiently derive neural cells. Additionally, this approach provides a platform for quantitative studies of molecular mechanisms that regulate neurogenesis of stem cells.

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Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells

Cited by 12 publications

References 66 publications

Self-regulatory factors of embryonic stem cells in co-culture with stromal cells enhance neural differentiation

Self-regulatory factors of embryonic stem cells in co-culture with stromal cells enhance neural differentiation

Cell‐Inspired All‐Aqueous Microfluidics: From Intracellular Liquid–Liquid Phase Separation toward Advanced Biomaterials

Stem cell colony interspacing effect on differentiation to neural cells

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