A quantitative, multi-national and multi-stakeholder assessment of barriers to the adoption of cell therapies

Cellular therapies, such as stem cell–based treatments, have been widely researched and numerous products and treatments have been developed. Despite this, there has been relatively limited use of these technologies in the healthcare sector. This study sought to investigate the perceived barriers to this more widespread adoption. An anonymous online questionnaire was developed, based on the findings of a pilot study. This was distributed to an audience of clinicians, researchers and commercial experts in 13 countries. The results were analysed for all respondents, and also sub-grouped by geographical region, and by profession of respondents. The results of the study showed that the most significant barrier was manufacturing, with other factors such as efficacy, regulation and cost-effectiveness being identified by the different groups. This study further demonstrates the need for these important issues to be addressed during the development of cellular therapies to enable more widespread adoption of these treatments

Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle.

<p>(<b>A</b>) Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. (<b>B–C</b>) Confocal images show Ad.MT58A-infected utricles (1×10⁹ TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C, 100 µm. (<b>D</b>) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 2×10⁸ TU/mL Ad.MT58A (1×M), and infection with 1×10⁹ TU/mL Ad.MT58A (5×M). (<b>E</b>) Confocal image of an Ad.MT58A-infected utricle (1×10⁹ TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5 DPV. Antibody labeling for BrdU and Ki-67 is shown in green and red, respectively. Scale bar, 100 µm. (<b>F</b>) Confocal image of an Ad.MT58A-infected utricle (1×10⁹ TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green and magenta, respectively. Scale bar, 100 µm. (<b>G</b>) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 µm. (<b>H</b>) Graph shows the mean number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the borders of the sensory epithelium in all panels.</p

Supporting cells that reenter the cell cycle after Ad.MT58A infection can progress to mitosis.

<p>(<b>A</b>) Confocal images show an Ad.MT58A-infected utricle (1×10⁹ TU/mL) that was fixed at 7 days post-virus (DPV) and co-labeled with antibodies to BrdU (red) and Ki-67 (green). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium. Scale bar, 100 µm. Insets show high-resolution views of nuclei in the sensory epithelium. Arrows indicate nuclei that labeled with antibodies to BrdU but not Ki67. Scale bar for insets, 5 µm. (<b>B</b>) Graph shows the mean number of BrdU-labeled nuclei (green data points) and Ki-67-labeled nuclei (red data points) per sensory epithelium at 5, 7, and 10 DPV. (<b>C</b>) Graph shows quantification of the percentage of the BrdU-positive population that did not label with Ki67 antibodies (green data points) and the percentage of the Ki-67-positive population that did not label with BrdU antibodies (red data points). (<b>D</b>) Confocal image of an adult mouse utricle infected with Ad.MT58A (1×10⁹ TU/mL) that was fixed at 7 DPV and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). (<b>E</b>) Confocal images of a utricle from an embryonic day 17.5 (E17.5) mouse that was fixed <i>in vivo</i> and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium, and arrows in D and E indicate PH3-Ser10/Ki-67 co-labeled nuclei. Scale bar for D–E, 100 µm. (<b>F</b>) Graph shows quantification of the percentage of the Ki-67-positive populations that labeled with antibodies to PH3-Ser10. The difference in the percentage of PH3-Ser10-positive/Ki-67-positive nuclei did not reach statistical significance (p>0.05; Student's t-test). The numbers above the gray bars indicate the mean number of PH3-Ser10-labeled nuclei per sensory epithelium. There were significantly more PH3-Ser10-positive nuclei in E17.5 utricles (p<0.05; Student's t-test).</p

Adenovirus primarily infects supporting cells but also some hair cells in adult mouse utricles <i>in vitro</i>.

<p>(A–D) Low magnification (20×/0.75 NA) confocal images of utricles from adult mice that were incubated with increasing concentrations of adenovirus engineered to express green fluorescent protein (GFP, green) under the control of a CMV promoter. Utricles were fixed and labeled 3 d after adenovirus was washed out. Hair cells are labeled with antibodies to myosin VIIA (magenta). (A′–D′) Fluorescent phalloidin labeling in the utricles from A–D. Scale bar for A–D', 100 µm. (E) High-resolution (63×/1.4 NA) confocal section taken at the apical surface of the sensory epithelium shows a cluster of GFP-positive supporting cells (green). The supporting cell apical surfaces have characteristic polygonal shapes compared to the circular profile of hair cells labeled with anti-myosin-VIIA (magenta). (F) View of a confocal image stack parallel to the apical-basal axis of supporting cells and hair cells shows a supporting cell expressing GFP (green), but its neighboring myosin-VIIA-labeled hair cell (magenta) does not. Scale bar, 5 µm. (G) Same view as in F shows a GFP-expressing hair cell. Scale bar, 5 µm. (H) A graph showing quantification of the percentage of GFP-expressing supporting cells (black circles) and hair cells (magenta circles) with increasing concentration of adenovirus. Myosin VIIA labeling was used to distinguish between supporting cells and hair cells, and sigmoidal equations (black and magenta lines) were fit to the data points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048704#pone-0048704-t001" target="_blank">Table1</a> for equation and coefficients).</p

Supporting cells that reenter the cell cycle in Ad.MT58A-infected utricles may be capable of differentiating towards a hair-cell-like fate.

<p>Shown are confocal images taken from Ad.MT58A-infected utricles (1×10⁹ TU/mL) fixed at 10 days post-virus (DPV) and labeled with antibodies to BrdU (green) and myosin VIIA (magenta). Growth medium was exchanged for differentiation medium at 5 DPV. (<b>A–B</b>) Confocal images show views parallel to the long axis of the hair cells. The arrowhead points to a BrdU-positive/myosin-VIIA-positive cell that does not extend to the apical surface and is probably damaged or dying. The other BrdU-positive/myosin-VIIA-positive cells extend from the hair nuclear layer up to the apical surface, where they appear to display tiny, bundle-like projections. BrdU-positive/myosin-VIIA-positive cells are located within close proximity to these hair-cell-like cells (arrows), suggesting a divisional pair. Scale bars, 5 µm. (<b>C–E</b>) Confocal images of three hair-cell-like cells at the level of the hair cell nuclear layer. Arrow in C indicates a neighboring cell that is BrdU-positive/myosin-VIIA-negative. Scale bars, 5 µm.</p

Coefficient values for sigmoidal curve fits.

<p>Coefficient values for sigmoidal curve fits.</p

Infection with Ad.MT58A is both necessary and sufficient for the observed proliferative response.

<p>(<b>A–D</b>) Confocal images of utricles that were individually infected with Ad.O, Ad.K, Ad.S, or Ad.MT58A and cultured in the presence of BrdU for 5 days post-virus (adenovirus concentrations equal to those used for co-infection experiments). Significant BrdU labeling (green) is only detected in the sensory epithelium of a utricle infected with Ad.MT58A. Hair cells are labeled with an antibody to myosin VIIA (purple). (<b>E–F</b>) Confocal images of utricles infected with 1×10⁹ TU/mL (5×) and 2×10⁹ TU/mL (10x) of Ad.MT58A. Inset in F shows a zoomed region of the sensory epithelium. Scale bar for inset, 20 µm. (<b>D'–F'</b>) Confocal images of the BrdU channel in D–F without the myosin VIIA overlay. White dashed lines demarcate the borders of the sensory epithelium. Scale bar for A–F', 100 µm. (<b>G</b>) Quantification of the number of BrdU-labeled nuclei per sensory epithelium for the different adenovirus combinations tested. O: Ad.O, K: Ad.K, S: Ad.S, M: Ad.MT58A, OKSM = co-infection with Ad.O, Ad.K, Ad.S, and Ad.M T58A. The difference in the number of BrdU-positive nuclei in co-infected utricles and utricles infected with Ad.MT58A did not reach statistical significance, but the increases in BrdU-positive nuclei at higher concentrations of Ad.MT58A were significant (asterisks indicate a significant difference from all other conditions; p<0.05; One-way ANOVA with Tukey's Test of Multiple Comparisons; n = 4 utricles).</p

BrdU-labeled cells in utricles co-infected with Ad.O, Ad.K, Ad.S, and Ad.MT58A are present after four weeks in culture and many appear in doublets.

<p>(A–B) High-resolution (63×/1.4 NA) views both orthogonal (A) and parallel to the apical-basal axis of supporting cells and hair cells in a co-infected utricle that was cultured for 7 days post-virus. BrdU was included in the culture medium for the entire period after virus washout. Hair cells and nuclei that have entered S-phase are labeled with antibodies to myosin VIIA (magenta) and BrdU (green), respectively. The two BrdU-positive nuclei in B appear to be a division pair, with one positioned at the level of hair cell nuclei and the other at the supporting cell nuclear layer. Scale bar in A, 20 µm. Scale bar in B, 5 µm. (C) Graph shows quantification of the mean number of BrdU-positive nuclei per sensory epithelium versus time in culture. Data from co-infected utricles are shown in gray, and data from control utricles infected with GFP are shown in black. (D) Graph shows the percentage of BrdU-positive nuclei that appeared as doublets in co-infected utricles (same utricles used for the gray data points in C). Subtracting the percentage from 100 yields the percentage of nuclei that appeared as singlets. (E) Confocal image of a co-infected utricle fixed at 8 days post-virus and labeled with antibodies to BrdU (green) and activated caspase 3 (red). Arrow points to a pyknotic nucleus that labeled with both antibodies. Scale bar, 10 µm.</p

Expression and localization of NgAgo.

<p><b>A</b>. Components of the three NgAgo proteins. <b>B</b>. Detection of HA- and Flag-tagged NgAgo by Western blots. <b>C</b>. Subcellular localization of NgAgo with one NLS signal sequence (Flag-tagged) and two NLS signal sequences (HA-tagged). Red signals were NgAgo (HA or Flag antibody stained, secondary antibody labeled by Alexa594). Nucleus was stained by DAPI and pseudocolored blue. Scale bar: 50 μm.</p

NgAgo showed no genome editing activity on plasmid and genomic DNA.

<p><b>A</b>. Untagged NgAgo produced green fluorescence positive cells independent of guide DNA. SaCas9, tagged NgAgo or untagged NgAgo were cotransfected with reporter plasmid DNA into HEK293T cells. In SaCas9 transfection single strand guide DNA was included to equate transfection conditions. Scale bar: 100 μm. <b>B</b>. Next-generation sequencing of the target region of reporter plasmid DNA amplified from cells co-transfected with untagged NgAgo and F2+R1 oligos. The most appearing seven types of reads are listed with their percentages. Positions of the guide oligos are highlighted, with direction indicated by arrows. <b>C</b>. Sequencing of the target region of human <i>MYC</i> promoter amplified from cells co-transfected with untagged NgAgo and the indicated oligos. The most appearing seven types of reads are listed with their percentages. Positions of the guide oligos are highlighted, with direction indicated by arrows.</p