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How to Identify Human Stem Cells in Animal Tissues

Stem cell therapy has advanced significantly over the last decade. In particular, it has shown promising results in regenerating cells in certain tissues of critical importance for life but limited natural ability to regenerate, including the central nervous system and myocardium.

Clinical trials involving stem cells are underway, and in each case, stem cell therapy must first be tested for efficacy and safety using preclinical animal models. This raises an important question: how can we distinguish injected or implanted human stem cells from the animal host’s own cells?

One option is to implant or inject human stem cells that have been genetically modified to express reporter genes. Some reporter gene products have the advantage of being detectable non-invasively in vivo, such as bioluminescence resulting from activity of the luciferase reporter gene (demonstrated here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191083/ and here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541295/). This modality can be extended to ex vivo imaging of fresh or frozen tissue sections for more detailed morphologic analysis of reporter gene expression. While the reporter gene imaging method has the potential to offer an exciting look at stem cell localization in vivo across multiple time points, it is time-consuming and costly to establish and validate each model and requires specialized imaging equipment. It may also be difficult to reconcile tissue collection, processing, and storage methods required for reporter gene analysis with those required for routine histopathology.    

As a practical alternative, immunohistochemistry (IHC) is a valuable and well established tool for identifying human cells in animal tissues. IHC can often be performed using formalin-fixed paraffin-embedded (FFPE) tissues as routinely processed for histopathologic interpretation. IHC offers easy detection of stem cell localization without compromising overall tissue morphology (demonstrated here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161450/). It also avoids potential alteration of stem cell function or host response as an unintended consequence of reporter gene introduction or the inability to detect stem cells due to the loss of reporter gene expression over time.

Some of the most promising commercially available human-specific antibodies with applications in preclinical stem cell therapy research include Ku80, anti-human nuclear protein, and anti-human mitochondria (hMito). These three antibodies can be used to identify xenotransplanted human stem cells regardless of lineage. The target of the Ku80 antibody is the 80-kilodalton subunit of the Ku heterodimer protein, which is also known as ATP-dependent DNA helicase II, an enzyme involved in repair of double stranded DNA breaks in the nucleus of human cells. Multiple anti-human nuclear protein antibody clones are available for use, with one of the most common versions binding histone H1, a protein that aids in structural stability and conformation of DNA in the nucleus. Similarly, there are multiple clones of hMito antibody available for use, which recognize various epitopes on or within human mitochondria, abundant energy producing organelles found in the cytoplasm of all cells.

Tissue-specific stem cell markers, such as STEM121, and STEM123, are also useful for identifying human stem cells in preclinical animal models using IHC. STEM121 binds to a human cytoplasmic protein found in brain, pancreas, and liver. STEM123 binds specifically with human glial fibrillary acidic protein (GFAP), which is found in astrocytes. Both of these markers are particularly useful for tracking human neural stem cells transplanted into an animal’s central nervous system.

Importantly, existing research demonstrates that these stem cell markers are stable and continue to be expressed even after cellular differentiation, making it possible to track xenotransplanted stem cells for an extended period of time after injection or implantation into an animal. These markers have been found not to cross-react with native proteins in rodents, cynomolgus monkeys, and pigs, which are common species used in preclinical stem cell studies.

Are you planning a preclinical assessment of stem cell therapy products? Consider incorporating IHC for clean and easy stem cell tracking.

HSRL Specializes in Histopathology & Specimen Storage Services

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