Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. (C) 2014 The Authors. Published by Elsevier B. V

Arrayed cellular environments for stem cells and regenerative medicine

The behavior and composition of both multipotent and pluripotent stem cell populations are exquisitely controlled by a complex, spatiotemporally variable interplay of physico-chemical, extracellular matrix, cell-cell interaction, and soluble factor cues that collectively define the stem cell niche. The push for stem cell-based regenerative medicine models and therapies has fuelled demands for increasingly accurate cellular environmental control and enhanced experimental throughput, driving an evolution of cell culture platforms away from conventional culture formats toward integrated systems. Arrayed cellular environments typically provide a set of discrete experimental elements with variation of one or several classes of stimuli across elements of the array. These are based on high-content/high-throughput detection, small sample volumes, and multiplexing of environments to increase experimental parameter space, and can be used to address a range of biological processes at the cell population, single-cell, or subcellular level. Arrayed cellular environments have the capability to provide an unprecedented understanding of the molecular and cellular events that underlie expansion and specification of stem cell and therapeutic cell populations, and thus generate successful regenerative medicine outcomes. This review focuses on recent key developments of arrayed cellular environments and their contribution and potential in stem cells and regenerative medicine

Concise review: Microfluidic technology platforms: Poised to accelerate development and translation of stem cell-derived therapies

Stem cells are a powerful resource for producing a variety of cell types with utility in clinically associated applications, including preclinical drug screening and development, disease and developmental modeling, and regenerative medicine. Regardless of the type of stem cell, substantial barriers to clinical translation still exist and must be overcome to realize full clinical potential. These barriers span processes including cell isolation, expansion, and differentiation; purification, quality control, and therapeutic efficacy and safety; and the economic viability of bioprocesses for production of functional cell products. Microfluidic systems have been developed for a myriad of biological applications and have the intrinsic capability of controlling and interrogating the cellular microenvironment with unrivalled precision; therefore, they have particular relevance to overcoming such barriers to translation. Development of microfluidic technologies increasingly utilizes stem cells, addresses stem cellrelevant biological phenomena, and aligns capabilities with translational challenges and goals. In this concise review, we describe how microfluidic technologies can contribute to the translation of stem cell research outcomes, and we provide an update on innovative research efforts in this area. This timely convergence of stem cell translational challenges and microfluidic capabilities means that there is now an opportunity for both disciplines to benefit from increased interaction

Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of Mesenchymal progenitor cells

Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications

Pearson’s correlation coefficients for paired chambers from individual MBA experiments.

<p>Pearson’s correlation coefficients for paired chambers from individual MBA experiments.</p

qPCR Primer Sequences.

<p>qPCR Primer Sequences.</p

Analysis of selected inhibitor concentrations on osteogenesis under standard conditions.

<p>A ELF97 (green) and PI (red) staining of MPCs treated with CHIR, IWP-4 and IWR-1 for 7 days. Scale bar, 100 µm. B Alizarin red staining of MPCs treated with combinations of CHIR, IWP-4 and IWR-1 for 21 days. Scale bar, 100 µm. C) RT-qPCR determination of expression of osteogenic marker genes after 7 days D) qPCR determination of expression of osteogenic markers genes after 21 days. RT-qPCR data is shown as mean±SEM. N = 3, p<0.05 (*), p<0.01 (**), p<0.001 (***).</p

Microfluidic screening reveals heparan sulfate enhances human mesenchymal stem cell growth by modulating fibroblast growth factor-2 transport

Cost-effective expansion of human mesenchymal stem/stromal cells (hMSCs) remains a key challenge for their widespread clinical deployment. Fibroblast growth factor-2 (FGF-2) is a key hMSC mitogen often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF-2, which critically interacts with cell surface heparan sulfate (HS). We assessed the interplay of FGF-2 with a heparan sulfate variant (HS8) engineered to bind FGF-2 and potenti-ate its activity. Bone marrow-derived hMSCs were screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 lg/ml) increased hMSC proliferation and cell number after 3 days, with an effect equivalent to FGF-2 (50 ng/ml). In combination, the effects of HS8 and FGF-2 were additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provided insights into modulation of FGF-2 transport by HS8. HS8 treatment induced proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF-2 accumulation, whereas responses to FGF-2 occurred primarily in the upstream chambers. Adding HS8 along with FGF-2, however, maximized the range of FGF-2 effectiveness. Measurements of FGF-2 in static cultures then revealed that this was because HS8 caused increased endogenous FGF-2 production and liberated FGF-2 from the cell surface into the super-natant. HS8 also sustained levels of supplemented FGF-2 available over 3 days. These results suggest HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF-2 production and maximizing the effect of supplemented FGF-2. This is an exciting strategy for cost-effective expansion of hMSCs

MBA screening of Wnt modulators in MPC osteogenesis.

<p>A Panel of screening conditions in MBAs. Numbers denote concentrations of the various molecules, in µM. B Confocal microscopy images of endpoint PI (DNA) and ELF97 (alkaline phosphatase activity) staining from a representative experiment. Direction of fluid flow was from top to bottom. C Heatmaps of expression indices (see Methods) for DNA, ELF97, and ELF97/DNA ratio. The average expression index of 2 runs from each of 2 MPC donors (4 in total) is shown, and units represent global standard deviations of difference relative to the global mean. For data from individual runs, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s002" target="_blank">Figs. S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s005" target="_blank">S5</a>. D Higher magnification fluorescence images of representative MPCs in MBA displaying alkaline phosphatase activity (ELF97) and DNA staining (PI). Scale bar: 200 µm. E Main effects plot showing effect of DONOR, CHIR99021 (CHIR), IWP-4, IWR-1 and POSITION on expression index for ELF97/DNA ratio. F Interaction effects plot showing effects of 2 combined factors on ELF97/DNA ratio.</p

Validation of MBA culture parameters and MPC seeding.

<p>A Comparison of cell morphology in 100 µm (top) versus 250 µm-high (bottom) devices. Scale bar, 200 µm. B Comparison of medium exchange regimes varied from conditions in top panel of A –10 µL/h flowrate (top) and periodic flow-stop (bottom). Scale bar, 200 µm. C Comparison of surface coating regimes varied from conditions in top panel of A – FBS-coated substrate (top) and collagen-I-coated substrate (bottom). Scale bar, 200 µm. D Phase contrast micrographs of MPCs in static plate controls and microbioreactor arrays in suspension directly after seeding, and attached after 4–5 h, just prior to the start of fluid flow. Scale bar, 200 µm. E Heatmap showing distribution of MPCs seeded into a MBA at representative experimental densities. F Graph showing average cells per chamber as a function of row. G Graph showing average cells per chamber as a function of column. H Live/dead staining of MPCs after 7 days. Scale bar, 100 µm.</p