Morula complementation restores fetal kidneys in xenocompatible SALL1 null sheep

The global shortage of donor organs has driven a massive push toward xenotransplantation—the transplantation of animal organs into humans. Current state-of-the-art approaches focus on "humanizing" livestock, such as pigs, by using CRISPR to knock out major xenoantigens (sugar molecules that trigger immediate immune rejection). However, even the most heavily edited porcine organs face persistent hurdles. These include physiological incompatibilities and the risk of transmitting animal pathogens.

A radical alternative is emerging: instead of making a pig organ look human, why not use the animal as a living bioreactor to grow actual human organs? This requires a sophisticated biological "shell game" known as embryo complementation. The goal is to create a host animal that is genetically incapable of developing a specific organ. This leaves an empty developmental niche that can be filled by donor cells. While this has been demonstrated in rodents and pigs, scaling this to large, clinically relevant animal models remains an open challenge.

The limitations of the porcine status quo

Most research in organogenesis complementation has focused on pigs. However, the field faces a bottleneck. Growing complex, human-scale organs in a host requires precise control over the "empty niche." This is the space where the target organ should be. If the host's own cells compete with the donor cells, the resulting organ may be malformed.

Furthermore, current xenotransplantation models rely on intensive multi-gene editing to prevent rejection. Recent studies show that reaching the "gold standard" of clinical readiness might require dozens of genomic edits. This increases the complexity and metabolic cost of producing these animals. There is a pressing need for a new model species. Such a species could provide a scalable platform for growing large, functional organs.

Engineering an empty renal niche

The researchers addressed this by turning to domestic sheep. They employed a multi-step genome engineering and cloning pipeline to create a specialized host. The process relies on three critical architectural choices:

1. Targeting the SALL1 master regulator: The team targeted the Spalt-like transcription factor 1 (SALL1) gene. This gene is essential for kidney development. They used Cas9-mediated editing to create two distinct host phenotypes. First, they used a single guide RNA (gRNA) to create a "hypomorph"—a version of the gene that still functions partially. This leads to hypoplasia (underdeveloped kidneys) [, Figure 2]. Second, they used dual gRNAs to delete a large 3.2 kb segment of exon 2. This effectively created a "null" allele that causes agenesis (the complete absence of kidney formation) .

2. Ensuring xenocompatibility: To prepare the host for potential human cell integration, the researchers simultaneously knocked out the CMAH and GGTA1 genes. These genes produce the primary xenoantigens that cause rapid rejection in humans.
3. Morula aggregation: Instead of injecting single cells into a blastocyst, the authors used "morula complementation." This involves aggregating two embryos at the morula stage (a solid ball of cells). This technique combines one edited TKO (triple knockout) host and one transgenic donor to maximize chimaerism.

Restoring the kidney through complementation

The effectiveness of this approach depends on the ability of donor cells to rebuild the missing organ. The authors used female donor cells carrying an mCherry fluorescent reporter to track donor-derived parts of the fetus.

The results are striking. In the chimaeric fetuses, the researchers found that donor cells successfully repopulated the empty kidney niche. The paper reports an average chimaerization efficiency of 54% across all complementation groups [Table 4]. This represents the percentage of fetuses that successfully integrated both host and donor cells. Most importantly, the kidneys derived from the donor cells were anatomically and histologically normal. Microscopic examination via H&E staining showed that these donor-derived kidneys possessed essential building blocks: glomeruli (filtering units), renal tubules, and a healthy population of stromal cells .

While the size of these rescued kidneys varied, they clearly outperformed the host's own capacity. In the case of the $SALL1$ null hosts, which should have had no kidneys, the donor cells filled the void. They produced functional renal structures where none should have existed.

Unresolved biological complexities

Several significant hurdles remain before this can move toward clinical application.

First, there is an issue with scaling and growth. The authors observe that while the kidneys were histologically normal, they did not consistently reach the average size of a wild-type male kidney. The rescued organs tended to fall within the size range of the donor or the host. They rarely achieved the full physiological scale required for a mature animal.

Second, the "chimaera" is not a perfect hybrid. The researchers note that certain structures derived from the ureteric bud remain a mix of host and donor cells. This includes the collecting ducts and the microvasculature (the tiny blood vessels). For a truly "humanized" organ, the entire vascular and ductal network would ideally need to be donor-derived.

Finally, the mortality rate in the $SALL1$ null (complete knockout) line was high. The paper reports that only 3% of the $CGS20null$ fetuses showed a heartbeat at the collection stage. This suggests that the total loss of $SALL1$ may have deleterious pleiotropic effects (side effects affecting other organ systems) that limit host viability.

The verdict: A new model for the factory of the future

This research provides a "yes" to whether sheep can serve as a viable large-animal model for in vivo organ generation. By successfully restoring kidney development in a $SALL1$-deficient host, the authors demonstrated that molecular mechanisms are conserved. These mechanisms are similar enough between rodents and ruminants to allow for successful complementation.

This moves the field from asking "can we make a chimaera?" to "how can we optimize the chimaera?" The transition from pigs to sheep offers a strategic shift. It could potentially bypass cultural and religious sensitivities associated with porcine models. It also provides a platform for growing human-scale organs. The next frontier will be refining the "niche." Scientists must ensure that not just the parenchyma (the functional tissue), but also the vital plumbing and blood supply, are entirely donor-derived.

Figures from the paper