How to: generate mouse-human chimeric embryos

A recent article published in Nature Protocols has detailed a breakthrough approach in generating mouse-human chimeric embryos with human pluripotent stem cells. This protocol may be applied to produce unprecedentedly realistic animal models for the study of human disease and development.

Chimeras are organisms comprised of genetically distinct cell lineages. Although rare in nature, laboratory-produced chimeras have advanced biomedical research by enabling the creation of humanised animal models. However, previous methods to generate robust mouse-human chimeric embryos have been unsuccessful. These approaches have involved injecting undifferentiated human pluripotent stem cells (hPSCs), or precursors of mature human cells, into mouse blastocysts.

If successful, chimeric embryos can be used to generate more realistic animal models of human disease and development. When applied in larger animals, artificially generating human cells, tissues and organs may even alleviate shortages in organ transplantation.

A recent paper described a novel protocol for generating human-mouse chimeras containing 0.14-4.06% human cells. As summarised in the Figure below, three main stages are involved. These include: conversion of hPSCs from a primed to naïve state (1-4), injection of naïve hPSCs into mouse blastocysts (5), and quantification of human cells in mouse-human chimeras (6). Below, we summarise each of these stages in more detail to help you generate human-mouse chimeric embryos.

Figure: Main stages for generating mouse-human chimeric embryos

Conversion of hPSCs from primed to naïve state

More developed, primed hPSCs are first converted into naïve, undifferentiated hPSCs before producing chimeric embryos. This is induced by transiently inhibiting mTOR. 2iLI medium then maintains hPSCs in an undifferentiated state. Since 2iLI medium is similar to the 2iL medium used to culture mouse embryonic stem cells (mESCs), hPSCs would behave like mESCs when later introduced into mouse blastocysts. This permits the robust generation of mouse-human chimeric embryos. 

The steps involved in this stage are:

1. Incubate primed hPSCs overnight with 10M ROCK inhibitor Y-27632 in hESC medium.
2. Incubate hPSCs for 3 hours in 2iLI medium with either 10M Torin1 or 10M rapamycin to transiently inhibit mTOR.
3. Dissociate hPSCs into single cells with trypsinisation.
4. Incubate primed hPSCs on mitomycin C-treated mouse embryonic fibroblast (MEF) feeders in fresh 2iLI medium, changing the medium daily.
5. After 3-4 days, pick out dome-shaped colonies that are large, bright and undifferentiated with a high nucleus-to-cytoplasm ratio. These are the successfully converted naïve hPSCs.
6. Dissociate colonies into single cells. Replate on MEF in 2iLl medium, termed passaging.
7. Repeat for ~10 passages to ensure naïve hPSC lines contain rapidly proliferating and few differentiated cells.
8. Incubate cell cultures at 37°C, replacing the medium daily and passaging cells every 3-4 days.

Follow these guidelines to ensure successful primed-to-naïve hPSC conversion:

• The use of hPSCs and animals should be approved by an institutional ethics review committee.
• Conversion should be conducted by a specialist experienced in culturing hPSCs and mESCs.
• Store freshly made 2iLI medium at 4ºC and use within 1 month.
• MEF cells must be plated at least 1 day and washed 3 times in an appropriate hPSC basal medium before their use as feeders.
• Culture hPSCs at physiological oxygen levels (5%) instead of atmospheric levels (21%).
• Restrict culturing time to a maximum of 2-3 months to maintain hPSCs in an undifferentiated and genomically stable state.
• Eliminate UV radiation sources in the cell culture room by replacing fluorescent with yellow light bulbs in cell culture hoods and installing covers to block UV from ceiling lights.
• Conduct routine PCR testing of cells for mycoplasma contamination.
• Wear a face mask in the cell culture room to reduce risk of spreading mycoplasma.
• Thaw and use a new vial of low passage hPSCs when the hPSC passage number reaches 30-40.

Injecting naïve hPSCs into mouse blastocysts

Trained specialists should be employed at this stage to inject hPSCs into mouse blastocysts and transfer blastocysts into pseudopregnant mice. During gestation, naïve hPSCs would co-develop with mESCs of mouse blastocysts. Mouse-human chimeric embryos are obtained at the E17.5 stage.

The steps involved in this stage are:

1. Label hPSCs with GFP-expressing lentiviruses and pick GFP+ colonies.
2. Passage colonies a few times to yield naïve hPSC cultures with 100% GFP+ colonies.
3. Dissociate GFP+ naïve hPSCs into single cells. Select round and bright cells for injection.
4. Inject 5-7 hPSC cells into mouse blastocysts.  
5. Transfer blastocysts into pseudopregnant mice within 1-2 hours after injection.
6. Retrieve mouse embryos at different developmental stages and analyse tissue sections with fluorescence imaging.

Quantifying human cells in mouse-human chimeric embryos

This final stage assesses the number of human cells in the resultant mouse-human chimeras. Next-generation sequencing (NGS) of the conserved V3 variable region of 18S rDNA is employed to accurately quantify human DNA. An 8 base-pair mismatch in the centre of human and mouse V3 regions allows for unambiguous sequence identification.

The steps involved in this stage are:

1. Extract DNA from E17.5 mouse-human chimeric embryos.
2. Prepare human and mouse genomic DNA standard curves to account for different 18S rDNA copy numbers in individual humans and mice.
3. PCR amplify mouse and human 18S rDNA V3 regions using barcoded primers.
4. Pool, purify, quantify and perform PhiX spike-in for 18s rDNA to generate a sequencing and perform NGS.
5. Perform bioinformatic quality control and demultiplexing of the sequenced reads.
6. Map the sequenced reads against human and mouse reference V3 sequences.
7. Calculate percentage of human DNA in total genomic DNA according to percentage of human reads in total reads.

Consider the following to optimise NGS and human 18s rDNA detection:

• Set up a pre-PCR amplification area with dedicated reagents and tools to avoid PCR contamination.
• Employ trained specialists for NGS and bioinformatic analysis.
• Prepare accurate human and mouse genomic DNA standards by using freshly purified human genomic DNA from the same human cell line and mouse genomic DNA from same strain used to make blastocysts.
• Human 18S rDNA is more reliably quantified in genomic DNA freshly isolated from embryos compared to DNA isolated from fixed and embedded embryos.

The generation of chimeras with this breakthrough protocol, as the authors of the paper said, “may enable many applications previously considered impossible”. Realising the potential of stem cell technology could give rise to a transformation in biomedical research.

Image credit: Freepik – Freepik