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Stem cell colony interspacing effect on differentiation to neural cells.

ABSTRACT: 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.

SUBMITTER: Joshi R

PROVIDER: S-EPMC6175657 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Stem cell colony interspacing effect on differentiation to neural cells.

Joshi Ramila R Fuller Brendan B Mosadegh Bobak B Tavana Hossein H

Journal of tissue engineering and regenerative medicine 20180813 10

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 ...[more]

PMID: 30058271

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Project description:High throughput gene expression analysis using qPCR is commonly used to identify molecular markers of complex cellular processes. However, statistical analysis of multi-dimensional, temporal gene expression data is complicated by limited biological replicates and large number of measurements. Moreover, many available statistical tools for analysis of time series data assume that the data sequence is static and does not evolve over time. With this assumption, the parameters used to model the time series are fixed and thus, can be estimated by pooling data together. However, in many cases, dynamic processes of biological systems involve abrupt changes at unknown time points, making the assumption of stationary time series break down. We addressed this problem using a combination of statistical methods including hierarchical clustering, change point detection, and multiple testing. We applied this multi-step method to multi-dimensional, temporal gene expression data that resulted from our study of colony size-dependent neural cell differentiation of stem cells. The gene expression data were time series as the observations were recorded sequentially over time. Hierarchical clustering segregated the genes into three distinct clusters based on their temporal expression profiles; change point detection identified specific time points at which the entire dataset was divided into several homogenous subsets to allow a separate analysis of each subset; and multiple testing procedure identified the differentially expressed genes in each cluster within each subset of data. We established that our multi-step approach pinpoints specific sets of genes that underlie colony size-mediated neural differentiation of stem cells and demonstrated its advantages over conventional parametric and non-parametric tests that do not take into account temporal dynamics of the data. Importantly, our proposed approach is broadly applicable to any multivariate data sets of limited sample size from high throughput and high content screening such as in drug and biomarker discovery studies.

Project description:Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation. Highlights • Induced pluripotent stem cells (iPSCs) were generated from MPS IIIA skin fibroblasts.• MPS IIIA iPSCs were successfully differentiated into neural progenitor cells (NPCs).• MPS IIIA NPC proliferation was reduced due to interference with FGF2 signalling.• Neurogenesis from MPS IIIA iPSC-derived NPCs is reduced via an unknown mechanism.

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Stem cell colony interspacing effect on differentiation to neural cells.

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Stem cell colony interspacing effect on differentiation to neural cells.

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