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Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells

Upinder Singh,R. Quintanilla,Scott Grecian,K. Gee,M. Rao,U. Lakshmipathy

Published 2012 in Stem Cell Reviews and Reports

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, placental-like and non-tissue specific forms. The non-tissue specific isozyme of AP is expressed in liver, bone and kidney. A similar isozyme was identified in pluripotent stem cells when monoclonal antibodies, TRA-2-49/6E, recognizing determinants of human embryonal carcinoma (EC) cells showed specific reactivity to this isoform.1 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 (iPSC).2–5 Although definitive measures of pluripotency involves in-vitro tri-lineage differentiation and in-vivo 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 TRA-1-60, TRA-1-81; AP; Oct4 and Nanog.6, 7 In the case of murine ESC, AP positive colony forming in-vitro assay is used as a measure of pluripotency to demonstrate the ability of cells to single cell clone, attach and proliferate.8 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 expression.9 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 efficiency.10 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 iPSCs.11 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 differentiation.12 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 colonies. Here 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 Sendai-virus based non-integrating reprogramming method.13 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 expansion.

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