Novel Live Alkaline Phosphatase Substrate for Iden

Authors: Upinder Singh Rene H Quintanilla Scott Grecian Kyle R Gee Mahendra S Rao Uma Lakshmipathy

Publish Date: 2012/03/18

Volume: 8, Issue: 3, Pages: 1021-1029

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Abstract

Alkaline phosphatases AP are a class of enzymes that hydrolyze phosphate containing molecules under alkaline conditions In humans there are primarily four different types of this enzyme intestinal placental placentallike and nontissue specific forms The nontissue specific isozyme of AP is expressed in liver bone and kidney A similar isozyme was identified in pluripotent stem cells when monoclonal antibodies TRA249/6E recognizing determinants of human embryonal carcinoma EC cells showed specific reactivity to this isoform1 AP is also known to be expressed at high levels in other pluripotent stem cell types such as embryonic germ cells EG embryonic stem cells ESC and induced pluripotent stem cells iPSC2 3 4 5 Although definitive measures of pluripotency involves invitro trilineage differentiation and invivo teratoma formation the most widely tested and validated panel for initial evaluation of ESC and iPSC consists of Stage Specific Embryonic Antigen SSEA4 Tumor Rejection Antigens TRA160 TRA181 AP Oct4 and Nanog6 7In the case of murine ESC AP positive colony forming invitro assay is used as a measure of pluripotency to demonstrate the ability of cells to single cell clone attach and proliferate8 A similar assay has been adapted for hESC where the sensitivity of the AP positive Colony forming assay to detect loss of pluripotent hESC has been found to be more sensitive than marker expression9 More recently the onset of AP positive colonies during early stages of reprogramming is used as an initial indicator of successful reprogramming of cells Furthermore in some instances the number of AP positive colonies is used as a mark of reprogramming efficiency10 Nevertheless this marker alone is not a definitive marker for the established iPSC clones Additional marker evaluation is necessary to identify and qualify bona fide iPSCs11 AP expression levels is a less sensitive measure to differentiate between undifferentiated and early differentiating cells since its expression level is reported to be varied depending on the lineage of differentiation12 AP staining has been used as a fast and easy method that results in a specific chromogenic or fluorescent staining of the pluripotent stem cells However the current methods using AP staining require cell fixation and/or result in end products that accumulate within the cells As a result these AP stained colonies often lose their morphology and cannot be propagated any further Inability to further culture selected pluripotent colonies identified using AP staining is a serious disadvantage of this methodology An ideal solution would be an AP substrate that stains cells without altering the integrity or characteristics of stem cells thereby allowing further expansion of the stained coloniesHere in we report the development and application of a novel fluorogenic live cell permeant substrate for AP Live AP Stain When incubated with cells for 20–30 min in basal culture media this stain shows specific and robust staining of pluripotent cells such as human EC murine and human ESC and iPSC with minimal or no staining of feeder cells and human fibroblasts Stained colonies retain their morphology and preserve their cell health The green fluorescence of the stained colonies is eliminated from cells 60–90 min after removal of the stain from the media We have further utilized this stain in iPSC work flow to identify emerging iPSC clones during reprogramming of BJ human fibroblasts using CytoTune™ a Sendaivirus based nonintegrating reprogramming method13 Clones with robust AP staining were manually picked and propagated further Expanded clones expressed other pluripotent markers differentiated into cell types representative of the three germ layers and maintained a normal karyotype These results indicate that AP Live Stain reported in this study does not alter the integrity or characteristics of the stained cells and is therefore an ideal tool to label early intermediates during iPSC generation or clonal populations of ESC for further selection and expansionFeederdependent human H9 ESC WA09 WiCell Research Institute and internally generated hiPSC were cultured in hESC media comprising of DMEM/F12 media containing 20 KnockOutTM SR KSR 10 µM MEM NonEssential Amino Acids solution 55 µM 2Mercaptoethanol and 4 ng/ml basic FGF on mitotically inactivated mouse embryonic fibroblast MEF feeder layer and maintained in a 5 CO2 37°C humidified incubator For feederfree culture hESC and hiPSC were cultured in StemPro® hESC SFM supplemented with 100 µM 2Mercaptoethanol and 8 ng/ml basic FGF grown on Geltrex® hESCqualified reduced growth factor basement membrane matrix coated onto tissue culture treated surfaces hESC and hiPSC were routinely passaged either using Collagenase IV or the StemPro® EZPassage™ disposable stem cell passaging tool hiPSC clones were mechanically picked and propagated during the early stages of reprogramming

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