Embryonic stem cell protocols frequently employ irradiated M

Embryonic stem cell protocols frequently employ irradiated MEFs as feeder cultures (Conner and fibroblast, 2001). We plated mouse embryonic stem (ES) PCI32765 either with or without feeders and compared ALP expression in both cultures using our multiplexed luminescence method. Higher levels of ALP were seen in ES cells plated alone or with feeders as compared with MEFs alone (Fig. 3, A). Feeder cells have only minor ALP expression and ALP level in MEFs was minimally detectable. Measurement of cell number with CTG following CDP and subsequent normalization (Fig. 3, B) could resolve each sample into 3 distinct groups. Remarkably, a two-fold increase in CTG signal in ES cells with feeders compared to ES cells suggests a very accurate measurement of viable cell numbers. The commonly used HTS quality control parameter Z′ was determined between ES cells and fibroblasts and between ES with feeders and fibroblasts (Z′=0.64 and 0.72, respectively). Z′ values>0.5 imply that such assay is perfectly applicable for HTS. In order to demonstrate a practical application of the multiplex assay for cell reprogramming, we seeded either 5 or 10 Mbd3/NuRD-depleted mouse fibroblasts cells in a 384-well plate and added doxycycline to initiate reprogramming by OKSM induction, as previously described (Rais et al., 2013). After 8days, we evaluated reprograming by scoring for Oct4-GFP reporter expression. The colonies were imaged by automated microscopy and images analyzed for GFP area and intensity (IMX score=Cell Area Average/Cell Intensity Average). Cells were then lysed and ALP expression was measured and normalized to CTG. ALP score is directly proportional to colonies size and number (ALP score=CDP/CTG) when comparing microscopy-derived images and our multiplex luminescence method (Fig. 4 A, B). ALP score may be more accurate than analysis of colonies made using image analysis, as the brightness of colonies can differ depending on levels of pluripotency marker expressed in a particular colony (Fig. 4, B). Microscopy images often have some artificial bright spots resulting from reflections from media or optical aberrations, which will require correction during image analysis. In contrast, the multiplex assay is based on ALP amount per is robust and limited in assay noise, and should limit false positive results. Forskolin is a well-known enhancer of reprogramming (Hanna et al., 2010) and treatment of WT mouse fibroblasts with Forskolin induced a 5-fold induction in the ALP score (Fig. 4, C). Therefore, our multiplexed ALP detection platform can be applied beyond the genetic induction of reprogramming and used in conjunction with small organic compound treatment of cells.
Discussion Stem cell research frequently monitors “stemness” during the reprogramming of somatic cells toward pluripotent or embryonic stem cells (Botman and Wyns, 2014). Previously established analysis of cell reprogramming has required genetic manipulation of common biomarkers to monitor stem cells - Oct4, Klf4, c-Myc, and Sox2 – as well as dedicated equipment such as high content microscopy and analytical software tools (Shi et al., 2008). However, since ALP production reflects cellular differentiation potential, it increases together with increasing pluripotency as well with number of pluripotent cells and therefore can be easily detected by multiplex assay (Fig. 2, A,B). Importantly, the multiplexed assay quantifies and qualifies ALP with colony size and number and can be used in time-course studies to monitor the temporal stages of reprogramming. Live-cell imaging using knock-in fluorescent transgenes such as Oct4-GFP can be more appropriate for time-course experiments, when a non-destructive approach is required. However, the simplicity of the method is applicable to multiple assay plates which can be processed appropriate time points, particularly when developing an assay for screening. Given that primary screens are usually “end point” procedures, we proposed our method as very efficient for HTS, where verification of hits can be accompanied by complementary imaging methods It should be noted that an end-point immuno-staining protocol will be significantly more labor-and reagent intensive and can thus be integrated as an orthogonal verification assay following a screening campaign for a cost-effective validation of activity. From the perspective of an automated HTS, normalization of ALP signal through cell viability will allow filtering of toxic compounds in at the level of the primary screen and quantify “reprogramming quality” of the hits such that resources are dedicated to productive chemical matter.