Alkaline Phosphatase Live Stain for early identification of induced pluripotent stem cells

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The accurate identification of pluripotent stem cells is especially critical during the process of somatic cell reprogramming, a complex multistep process that takes several weeks. Identifying true pluripotent colonies in a bed of transformed fibroblasts and other cell debris may be relatively straightforward to a trained eye but can be quite challenging in the absence of extensive experience with these cells.

Here we demonstrate an easy-to-use reprogramming method for generating induced pluripotent stem cells (iPSCs) from human somatic cells, as well as a novel cell staining protocol that simplifies early iPSC identification. Used together, these methods facilitate the transition from small-scale stem cell experiments to large-scale production of iPSCs for disease modeling and drug development.

Generate iPSCs using CytoTune®-iPS Sendai reprogramming

The CytoTune®-iPS Sendai Reprogramming Kit provides a highly efficient method for reprogramming human somatic cells—including fibroblasts (Figure 1), peripheral blood mononuclear cells (PBMCs), and CD34-positive blood cells—to generate iPSCs. This kit utilizes four nonintegrating Sendai viruses encoding Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, which have been shown to be critical for iPSC generation [1]. Sendai viruses replicate in the cytoplasm of infected cells. These ssRNA viruses do not require nuclear entry for transcription nor do they go through a DNA intermediate, thereby minimizing the possibility of transgene integration in the host cell. Lentivirus and other virus types commonly used for reprogramming contain transgenes that can integrate into the host genome, leading to unpredictable results. The CytoTune®-iPS Sendai Reprogramming Kit generates iPSCs with an efficiency as high as 1%, a significant improvement over standard lentivirus-based methods, which produce iPSCs with an efficiency of 0.0001% to 0.01%.

Figure 1. Emerging iPSCs generated using the CytoTune®-iPS Sendai Reprogramming Kit. BJ human fibroblasts (ATCC) were transduced overnight using the CytoTune®-iPS Sendai Reprogramming Kit , and culture medium was replaced the next day. One week post-transduction, the cells were seeded onto inactivated MEF feeder cells in human PSC medium (DMEM/F-12 containing 20% KnockOut™ Serum Replacement and 4 ng/mL bFGF). At 14 days post-transduction, emerging iPSCs were analyzed for alkaline phosphatase activity using the Alkaline Phosphatase Live Stain, and images were collected on a Zeiss® Axiovert® microscope using a 10x objective: (A) phase-contrast image, (B) fluorescence image (using FITC optical filters), (C) merged images.

Identify iPSCs with Alkaline Phosphatase Live Stain

During a reprogramming protocol, candidate iPSC colonies are generally examined for the expression of pluripotent markers using immunocytochemical analyses. The most common method for identifying iPSC colonies is the use of stem cell–specific antibodies, such as those that recognize SSEA4, TRA-1-60, and TRA-1-80 proteins, which are highly expressed on the surface of human stem cells. This method, however, is typically used after colonies emerge and therefore cannot be employed early in the reprogramming process to monitor colonies on the master plate.

Alkaline phosphatase (AP) activity has also been shown to be up-regulated in pluripotent stem cells, including undifferentiated embryonic stem cells (ESCs), embryonic germ cells (EGCs), and iPSCs. AP activity is often used to distinguish stem cells from feeder cells as well as from parental cells in reprogramming experiments. In contrast to antibody-based analyses, AP substrates can be used for early screening. These reagents, however, are often toxic to cells and therefore require endpoint analyses, sacrificing iPSCs in order to determine which colonies should be selected for further culture.

Unlike traditional AP stains, the Molecular Probes® Alkaline Phosphatase Live Stain enables live-cell labeling of pluripotent stem cells. When enzymatically turned over, this cell-permeant AP substrate produces a bright green-fluorescent product that then diffuses out of the cell, leaving behind no significant chemical or biological footprint [2]. In contrast to other AP stains, neither the Alkaline Phosphatase Live Stain nor its enzymatic product accumulates in the cell. Alkaline Phosphatase Live Stain can be used to identify emerging iPSCs via robust fluorescent staining of the colonies as early as 14 days post-transduction (Figure 1), and to select iPSC colonies for expansion at 21 days post-transduction. Alkaline Phosphatase Live Stain is manufactured to be free of bioburden and low in endotoxins. Importantly, it is nontoxic to emerging iPSCs and can therefore be used directly on master reprogramming plates to help identify colonies for further propagation [2]. Because the fluorescent signal is transient, Alkaline Phosphatase Live Stain can be used multiple times throughout the iPSC expansion process, as well as on established PSCs; the signal within the cells is not significantly detectable 2 hr after cell application (Figure 2).

Transient staining with the Alkaline Phosphatase Live Stain

Figure 2. Transient staining with the Alkaline Phosphatase Live Stain. A 500X stock solution of Alkaline Phosphatase Live Stain was diluted in DMEM/F-12 medium and directly applied to an adherent cell culture of H9 embryonic stem cells, following removal of growth medium. Cells were incubated with substrate for 20–30 min, gently washed three times, and then imaged in fresh DMEM/F-12 medium within 30 min of staining (A, B, C) and 2 hr post-staining (D, E, F). Images were collected on a Zeiss® Axiovert® microscope using a 5x objective: (A, D) phase-contrast images, (B, E) fluorescence images (using FITC optical filters), and (C, F) merged images. At 2 hr post-staining, the fluorescent signal within the cells is not significantly detectable as the substrate is turned over and the fluorescent product diffuses into the surrounding medium. Following visualization and/or manual selection, the cells were returned to normal culture conditions in fresh medium. Similar results are seen with iPSCs [2].

Confirm pluripotency with stem cell–specific antibodies

The pluripotency of iPSC colonies that stain positive for alkaline phosphatase activity can be confirmed using stem cell–specific antibodies. For example, colonies that are initially identified and selected using Alkaline Phosphatase Live Stain at the time of picking continue to express the membrane-surface markers SSEA4 and TRA-1-81 upon further propagation and expansion (Figure 3). Confirmed iPSC colonies are typically then expanded, banked, karyotyped, and analyzed for differentiation potential.

Figure 3. Confirmation of iPSC colony formation using immunocytochemical analysis. iPSCs generated with the CytoTune®-iPS Sendai Reprogramming Kit and selected at day 21 using Alkaline Phosphatase Live Stain were further expanded and then probed for pluripotent surface markers by labeling nonfixed cells with stem cell–specific antibodies. All incubations and washes were performed at 37°C with prewarmed DMEM/F-12 medium in order to maintain cell viability and normal live-cell morphology. A single colony that tested positive for alkaline phosphatase activity was (A) visualized using phase contrast, (B) labeled with anti–TRA-1-81 mouse monoclonal antibody followed by Alexa Fluor® 488 goat anti–mouse IgG secondary antibody, and (C) further labeled with Alexa Fluor® 647 anti-SSEA4 mouse monoclonal antibody. (D) The merged image shows multiplexed stem cell–specific labeling. Images were collected on a Zeiss® Axiovert® microscope using a 10x objective

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