Dielectrophoresis: A Review of Applications for Stem Cell Research

Dielectrophoresis can discriminate distinct cellular identities in heterogeneous populations, and monitor cell state changes associated with activation and clonal expansion, apoptosis, and necrosis, without the need for biochemical labels. Demonstrated capabilities include the enrichment of haematopoetic stem cells from bone marrow and peripheral blood, and adult stem cells from adipose tissue. Recent research suggests that this technique can predict the ultimate fate of neural stem cells after differentiation before the appearance of specific cell-surface proteins. This review summarises the properties of cells that contribute to their dielectrophoretic behaviour, and their relevance to stem cell research and translational applications

Dielectrophoresis based discrimination of human embryonic stem cells from differentiating derivatives

Assessment of the dielectrophoresis(DEP) cross-over frequency (f xo), cell diameter, and derivative membranecapacitance (C m) values for a group of undifferentiated human embryonic stem cell (hESC) lines (H1, H9, RCM1, RH1), and for a transgenic subclone of H1 (T8) revealed that hESC lines could not be discriminated on their mean f xo and C m values, the latter of which ranged from 14 to 20 mF/m2. Differentiation of H1 and H9 to a mesenchymal stem cell-like phenotype resulted in similar significant increases in mean C m values to 41–49 mF/m2 in both lines (p < 0.0001). BMP4-induced differentiation of RCM1 to a trophoblast cell-like phenotype also resulted in a distinct and significant increase in mean C m value to 28 mF/m2 (p < 0.0001). The progressive transition to a higher membranecapacitance was also evident after each passage of cell culture as H9 cells transitioned to a mesenchymal stem cell-like state induced by growth on a substrate of hyaluronan. These findings confirm the existence of distinctive parameters between undifferentiated and differentiating cells on which future application of dielectrophoresis in the context of hESC manufacturing can be based

A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture

AbstractMultiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting Gαs-, Gα-i/o- and Gα-q/11-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of Gα-q/11 as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a Gα-q/11 subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture

Paracrine signalling events in embryonic stem cell renewal mediated by affinity targeted nanoparticles

AbstractStem cell growth and differentiation is controlled by intrinsic and extrinsic factors. The latter includes growth factors, which are conventionally supplied in vitro in media exchanged daily. Here, we illustrate the use of affinity targeted biodegradable nanoparticles to mediate paracrine stimulation as an alternative approach to sustain the growth and pluripotency of mouse embryonic stem cells. Leukaemia Inhibitory Factor (LIF) was encapsulated in biodegradable nanoparticles and targeted to the cell surface using an antibody to the oligosaccharide antigen SSEA-1. Sustained release of LIF from nanoparticles composed of a solid Poly(lactide-co-glycolic acid) polyester or a hydrogel-based liposomal system, we term Nanolipogel, replenished once after each cell passage, proved as effective as daily replenishment with soluble LIF for maintenance of pluripotency after 5 passages using 104-fold less LIF. Our study constitutes an alternative paradigm for stem cell culture, providing dynamic microenvironmental control of extrinsic bioactive factors benefiting stem cell manufacturing

Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Elasticity of Human Embryonic Stem Cells as Determined by Atomic Force Microscopy

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young's modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young's modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs

Model of the relationship between epigenetically-regulated hESC biomarkers and the pluripotency transcription system.

<p>Changes in the expression of an epigenetically-regulated transcriptional regulator (e.g. HMGA1, GLIS2, PFDN5) achieved by siRNA transfection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref001" target="_blank">1</a>], or changes in associated CGI methylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref006" target="_blank">6</a>], feed through the cellular transcription network, resulting in a reduction in core pluripotency transcription factors (OCT4, NANOG, SOX2, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref002" target="_blank">2</a>]) and differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref003" target="_blank">3</a>]. The core pluripotency transcription factors are permanently downregulated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref004" target="_blank">4</a>], and epigenetic changes to gene promoters, CGIs and other regulatory regions of the genome occur to confer stability on the differentiated phenotype and prevent reversion to pluripotency or quasi-pluripotency [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref005" target="_blank">5</a>]. These changes confer permanent changes to the expression of the epigenetically regulated transcriptional regulators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref006" target="_blank">6</a>], and thus stabilise the differentiated phenotype [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.ref007" target="_blank">7</a>].</p

Epigenetically-defined hESC biomarkers have a role in pluripotency.

<p>(A) Transcriptional activators (Expressed hESC-UnMe-GA-CGIs) and transcriptional repressors (Expressed hESC-Me-GA-CGIs) identified as functionally overrepresented hESC biomarkers. (B, C) Functional testing of transcriptional regulators in RH1 hESCs by siRNA knockdown. (B) RT-qPCR data showing log₁₀ fold change in expression of the siRNA-targeted gene, and associated effects on OCT4, NANOG and SOX2. Changes are relative to GAPDH expression, normalised to RH1 hESCs treated with negative control siRNA IDS-NULL. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ND: Not Detected, even at 40 cycles of PCR. (C) Immunohistochemistry for NANOG and OCT4 72 hours after siRNA treatment. Scale bar = 100 μm.</p