Rational design and development was utilized to develop a novel nontoxic AP Live Stain. An ideal nontoxic fluorescent end product was first chosen followed by its phosphorylation to create a phosphatase substrate. Subsequent modifications were carried out to enhance the cell permeability of the phosphatase substrate to create few candidate molecules. The methods, structures and compositions of these molecules are described in a separate study (Manuscript under preparation). A modified phosphorylated green fluorescent dye referred to as “AP Live Stain” was evaluated for pluripotent stem cell detection in this study.
H9 ESC on feeders was stained with two available commercial products along with the AP Live Stain. Enzyme-labeled Fluorescence 97 (ELF-97) is a soluble phosphorylated molecule that yields green fluorescent precipitate when cleaved by AP. These resulting fluorescent precipitates can be visualized under FITC excitation/emission filter systems using standard fluorescence microscope. The staining is specific to the ESC colony with little or no signal in the surrounding feeder cells (Fig. ). Vector® Red yields a highly fluorescent bright red precipitate when cleaved by AP and can be viewed visually or with Texas Red® or Rhodamine excitation/emission filter systems. The staining is bright red, specific to the ESC colony with minimal background (Fig. ). Both these reagents are fast and simple to use but require permeabilization and fixation of the cells. The resulting stained colonies lose their integrity and peel off the dish with time, a sign of cellular mortality. Thus, AP assay using these reagents is an end-point assay where cells once stained cannot be expanded further for downstream applications. Live AP Stain on the other hand generates a soluble phosphorylated molecule inside the cell that in presence of AP yields a non-toxic green fluorescent product. This product can be visualized using FITC excitation and emission filter systems. ESC colonies show specific and robust staining while the surrounding MEF feeder cells remain unstained (Fig. ). This differs from the ELF-97 and Vector® Red AP stains since the fluorescent product from AP Live Stain does not accumulate in the cells and the stained cells loose the fluorescence as the fluorescent product permeates out of the cells with time.
Fig. 1 Traditional dyes that are used as substrates to measure elevated alkaline phosphatase activity in pluripotent stem cells, such as ELF97 (a) and Vector Red (b) result in stained cells that cannot be propagated further. Rational design and synthesis was (more ...)
Different types of cells were stained with the AP Live Stain to further demonstrate the specificity of the AP Live Stain. BJ human fibroblast is widely used as the parental cell type for somatic reprogramming using various methods. These cells demonstrated insignificant staining that required long exposure times (Fig. , panel A). MEF cells that are commonly used as feeders for pluripotent stem cell in a feeder-dependent culture also exhibited weak staining similar to human fibroblasts (Fig. , panel B). On the other hand, the human embryonal carcinoma cell line NTERA2 that is known to have gene expression profiles similar to embryonic stem cells1
stained positively with the AP Live Stain (Fig. , panel D)
Fig. 2 The AP Live stain was incubated with different cell types under identical conditions. Phase contrast and fluorescence images captured using FITC filter were captured using an Axiovert fluorescence microscope. Adobe Photoshop was used to generate overlap (more ...)
Several different types of stem cells demonstrated robust and specific staining with AP Live Stain under identical incubation conditions. Incubating cultured colonies of 129/SvEv mESC (Fig. ) or H9 hESC (Fig. ) on a layer of MEF feeder cells displayed robust and specific staining of mESC or H9 hESC over MEF feeder cells. Furthermore, incubating established iPSC lines cultures on feeders that are derived from BJ fibroblasts using CytoTune™ iPS-Sendai Reprogramming method show equally strong AP staining (Fig. ). The minor differences noted in intensities between different cells types is largely due to size of the colony with the signal more robust in the larger human ESC and iPSC relative to the smaller mouse ESC or monolayer embryonal carcinoma cells. Additionally, in all cases, the typical morphology of the ESC colonies with high nucleus to cytoplasmic ratio with distinct refractive colony edges were retained after staining with AP Live Stain and was similar to the unstained control.
The ability of AP Live stained cells to preserve their cellular integrity was further confirmed using PrestoBlue™ Cell Viability Reagent. Metabolically active cells continuously convert the PrestoBlue™ Reagent, from a blue compound with no intrinsic fluorescent value to a red product that is highly fluorescent, thereby increasing the overall fluorescence intensity (at 590 nm) of the media surrounding the cells. Non-viable cells, on the other hand, cannot metabolize the indicator stain resulting in no change in fluorescent intensity at 590 nm. The health of the cell under investigation can therefore be monitored by the change in the fluorescence intensity at 590 nm of the media. H9 ESC on feeders (Fig. , panel i) were stained with independent preparations of AP Live Stain to confirm pluripotent specific staining (Fig. , panels ii–viii). Wells with feeder-free H9 ESC samples were used to assess cell health using PrestoBlue™ cell vitality assay and the results represented as bar graphs (Fig. ). Control wells with cells that were not treated with AP Live Stain showed high levels of fluorescence indicating a large number of the cells to be metabolically active. Comparable values were obtained with all samples treated with the AP Live Stain. Statistical comparison between the AP Live stained cells to the positive control showed no significant difference. In contrast, the negative control groups (cells treated with known cell disruptive agents such as PFA and DMSO) showed significantly lower levels of fluorescence indicating significantly lower number of metabolically active cells. In comparison to the control, the difference was statistically significant with p values >0.005, as measured by ANOVA (represented as stars over the bars). These results collectively indicate that AP Live Stain does not alter integrity of stained cells.
Fig. 3 a Feeder-dependent H9 ESC cultures (i) were stained with 7 independent preparations of AP Live Stain (ii–viii). Images were collected at 10X magnification following 30 min incubation with AP Live Stain followed by washes. Specific staining (more ...)
The utility of AP Live Stain was further evaluated in reprogramming work flow. BJ human fibroblasts transduced with the CytoTune™ iPSC reprogramming particles resulted in formation of colonies 2 to 3 weeks post transduction. Emerging colonies (Fig. , panel i) stained positive with Live AP Stain (Fig. , panel ii), and were also positive for another pluripotent marker Tra-1-60 (Fig. , panel iii). 12 clones that stained positive with AP Live Stain were manually picked and seeded on to fresh dishes with MEF feeders. All of these clones were further expanded to passage 3 at which time six clones were further treated with AP Live Stain.
Fig. 4 a BJ fibroblasts transduced with CytoTune™ reprogramming particles show emergence of colonies that stain positive for AP Live Stain. These colonies also express the pluripotent marker, TRA-1-60. b 3 weeks after transduction, the emerging (more ...)
Figure (panel i) shows a representative image of one of these clones stained with the AP Live Stain. This positively stained clone was scored using a 27 gauge needle during which process parts of the cells around the scoring region was removed and images immediately captured to demonstrate that the iPSC clones labeled with AP Live stain colony had distinct scores with bare regions along the scored edges (Fig. , panel ii). Once the AP Live Stain was removed from the media and the cells allowed to recover for 2 h in normal ESC growth media, the scored colony was again imaged to show loss of the green fluorescence (Fig. , panel iii). These cells, when allowed to recover over 48 h, were able to propagate and phase contrast image of the scored colony shows that the gaps along the scored edges of the colony are now filled with proliferating cells (Fig. , panel iv). As a further confirmation of maintenance of pluripotency in the proliferating areas of the scored clone, all cells within the colony continued to stain positive for the pluripotence surface markers SSEA4 (labeled Green, Fig. , panel v) and TRA-1-60 (labeled Red, Fig. , panel v).
Three clones from the above experiment were further propagated to over passage 10 and periodically checked for expression of characteristic pluripotent markers (Fig. ): AP Live Stain (panel i), SSEA4 (panel ii), TRA-1-60 (panel iii), Oct 4 (panel iv) and Nanog (panel v). These clones were randomly induced to differentiate via embryoid body formation resulting in various cell types (Fig. ). The differentiated cell types stained positively for beta III tubulin an ectoderm neuronal marker (i), alpha feto protein, an endodermal liver marker (ii) and smooth muscle actin, a mesoderm marker. The clone at passage 12 continued to maintain a normal karyotype as determined by G-banding (Fig. ). Parallel H9 ESC cultures and iPSC clones that were not treated with AP Live Stain and maintained under similar culture conditions behaved identically to the above clones suggesting no long term impact of AP Live Stain on the cell characteristics.
Fig. 5 Of the 12 clones initially picked, 3 clones were further expanded and characterized. All clones expressed pluripotent markers, had tri-lineage differentiation potential and maintained a normal karyotype a A representative iPSC clone that was initially (more ...)
These results collectively demonstrate that the AP Live Stain is an inexpensive, straightforward and undemanding alternative to existing AP dyes with the added advantage of preserving cell integrity that allows for cell manipulation and proliferation post staining.