Results: 5

1.
Fig. 1

Fig. 1. From: Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells.

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 carried out to develop a AP Live Stain and shown to specifically stain pluripotent stem cells (c). The morphology of the fluorescent labeled cells appeared intact in comparison to cells stained with traditional dyes

Upinder Singh, et al. Stem Cell Rev. 2012 September;8(3):1021-1029.
2.
Fig. 2

Fig. 2. From: Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells.

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 images of phase contrast and fluorescence images. a BJ human Fibroblast, the most commonly used parental cell line for somatic reprogramming b Murine Embryonic Fibroblasts, mitotically inactivated, and commonly used as feeder cells for ESC and iPSC culture c 129/SvEv murine ESC cultured on inactivated MEF feeder cells d NTERA2 c.D1 human embryonal carcinoma cells e Human H9 (WA09) ESC cells cultured on inactivated MEF feeder cells f iPSC derived from BJ human fibroblast using CytoTune™ Reprogramming and cultured on inactivated MEF feeder cells

Upinder Singh, et al. Stem Cell Rev. 2012 September;8(3):1021-1029.
3.
Fig. 4

Fig. 4. From: Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells.

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 colonies were stained with AP Live Stain and colonies that showed robust fluorescent staining were manually passaged onto feeders for further expansion and characterization. At passage 3, the expanding clones showed distinct ESC-like morphology and stained positive for AP Live Stain (i). The stained colony was scored with a 27 gauge needle and parts of the colony removed. At this stage the scored colonies fluoresce green (ii). 2 h after staining with AP Live Stain and removal of the stain from the growth media, the scored cells do not show any fluorescence (iii). Following recovery of the AP Live stained and scored iPSC colonies for 48 h at 37 C, the scored areas are covered with expanding cells (iv) and the restored areas stained positive for pluripotent surface markers SSEA4 (green) and TRA-1-60 (red) (v)

Upinder Singh, et al. Stem Cell Rev. 2012 September;8(3):1021-1029.
4.
Fig. 5

Fig. 5. From: Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells.

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 picked based on robust AP Live staining was expanded to passage 10 under feeder-dependent conditions express pluripotent markers AP (i), SSEA4 (ii), TRA-1-60 (iii), Oct4 (iv) and Nanog (v). b Representative iPSC clone was differentiated via random embryoid body formation and at the end of 21 days of differentiation, resulting cells stained positive for lineage specific markers representative of the three germ layers, beta III Tubulin—ectoderm (i), Alpha feto protein—endoderm (ii) and Smooth muscle actin—mesoderm (iii). c Cytogenetic analysis performed on twenty G-banded metaphase cells from a representative iPSC clone demonstrated an apparently normal male karyotype (46, XY) and no abnormal cells were detected

Upinder Singh, et al. Stem Cell Rev. 2012 September;8(3):1021-1029.
5.
Fig. 3

Fig. 3. From: Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells.

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 of the ESC colonies was observed with all the samples. b Cell health of AP Live stained cells was assessed using PrestoBlue™ cell vitality dye. H9 ESC was seeded in 96 wells with 6 replicates for each test conditions. Wells were stained with 7 independent preparations of AP Live Stain. Wells not treated with AP Live Stain served as positive controls while wells treated with known cell disrupting agents such as Paraformaldeyhe (PFA) and Dimethyl sulfoxide (DMSO) served as negative controls. Two hour after staining, PrestoBlue™ was added to the wells and incubated for 20 h. At the end of the incubation period, fluorescence at 590 nm was measured and the average values obtained for each sample plotted as a bar graph with error bars representing standard deviation. * indicates conditions where the values obtained was statistically significant with P values >0.05, relative to Control

Upinder Singh, et al. Stem Cell Rev. 2012 September;8(3):1021-1029.

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