Odd cell out: detecting donor-derived macrophages in the brains of hematopoietic stem cell transplant recipients

From the Loeb Lab, Translational Science and Therapeutics Division

Where do they come from?
Where do they go?
Where do they come from… donor bone marrow-derived brain macrophages?

My version may not be as catchy as the original, but it does illustrate a question that Dr. Keith Loeb, an associate professor in the Fred Hutch Translational Science and Therapeutics (TST) Division, spends a lot of time thinking about. Dr. Loeb is an experimental hematopathologist—he splits his time between the clinic, where he diagnoses complications of blood stem cell transplantation therapies, and the lab, where he studies basic cancer biology. In a recent study published in Blood Advances, the Loeb Lab marries these two interests in their investigation of whether donor-derived blood stem cells can differentiate and engraft into the brain of transplant recipients, and what this might mean for the development of new, brain-targeted cell therapies.

Regarding donor-derived macrophages and the first question above, we already have an answer—we put them there. Or rather, doctors transplanted donor macrophage precursor cells (stem cells) into the bone marrow of a recipient as part of a hematopoietic stem cell transplantation (HSCT) procedure. HSCT therapy—pioneered at the Hutch in the 1950’s by Nobel Laureate Dr. E. Donnall Thomas and others—is now a common treatment for patients with life-threatening blood cancers or other hematological diseases. The treatment works by using chemotherapies or radiation to ablate a patient’s malignant or otherwise malfunctioning bone marrow stem cells and then replace them with those of a donor—often, this donor is an unrelated but biologically compatible individual. Once transplanted, donor stem cells provide the patient with functional hematopoietic precursors and a new immune system which can effectively fight off residual cancer (this is called the ‘graft-versus-tumor-effect’ for those interested).

However, donor cells also do what stem cells are programmed to do: they differentiate and colonize different parts of the host’s body. Exactly where they end up, though, is much less understood. One particular destination which has attracted considerable interest is the brain—several studies, primarily using mouse models, have found evidence that donor stem cells can engraft in host brain tissue, where they differentiate into a type of macrophage called a bone marrow-derived macrophage (BMDM). Data demonstrating this possibility in humans, however, is sparse (and important to have, since mice aren’t always representative model systems!). Thus, Dr. Loeb and colleagues set out to determine whether they could detect engraftment of donor BMDMs in HSCT patient brain tissue.

Immediately, the team faced the challenge of detecting comparatively rare donor-derived BMDMs in poorly-preserved, sometimes decades-old brain tissue specimens from HSCT recipients—think of it like trying to find a needle in a haystack while wearing oven mitts. This challenge necessitated two key innovations. First, the team only used specimens from female patients who received cells from a male donor. This allowed them to identify donor cells by staining the tissues with fluorescent probes against the X and Y chromosomes: donor cells would stain for both X and Y, while recipient cells would only stain for X. Second, Dr. Loeb and colleagues adapted commercially available reagents into a novel workflow which allowed them to simultaneously probe tissue samples for XY and IBA1, a protein marker for BMDMs and microglia. Cells which stained for both XY and IBA1 could therefore be conclusively identified as donor derived BMDMs.

Applying this staining methodology to tissue from 19 different patients and using custom software to automate the image analysis, the team was successful in their quest: on average, 8.4% of IBA1-positive brain microglia were donor derived in their cohort of HSCT patients, confirming that donor-derived stem cells can engraft in the brain following HSCT therapy in humans. Furthermore, the team found more donor derived BMDMs in tissue from patients who received multiple transplants compared to those who received a single transplant, and in patients who received myeloablative pre-transplant conditioning (this is the chemotherapy/radiation which destroys host stem cells to make room for the donor cells) compared to those who did not. Additionally, the number of donor derived BMDMs was greatest in patients with the longest post-transplant survival, suggesting that the donor cells are a stable entity in the brains of transplant patients.

Fluorescent microscopy staining depicting spots of green and red staining on a mostly dark background of faint green brain tissue
Frontal cortex biopsies from four female patients stained with probes against the X chromosome (green puncta), and the Y chromosome (red puncta) show male donor cells (white arrows). Nuclei are stained blue and tissue autofluorescence comprises the light green background signal. Image taken from publication.

Why should we care whether donor-derived stem cells can engraft in the brain following HSCT therapy? Well, because it’s cool. But also, because it may represent a creative method of delivering cellular treatments against neurological diseases to the brain—a notoriously difficult organ to target owing to the blood-brain barrier. “Our study, which represents the most comprehensive look at donor-derived BMDM engraftment in HSCT patients to date, provides proof-of-concept for these kinds of therapies in humans,” explains Dr. Loeb. “In addition, we identify transplant frequency, post-transplant survival, and pre-transplant conditioning as variables affecting engraftment, which could help optimize this procedure to eventually treat neurological disorders like Alzheimer’s with state-of-the-art microglia replacement therapies using bone marrow transplants.” And with diseases like Alzheimer’s and Parkinson’s still eluding scientists worldwide, it’s going to take creativity to make meaningful progress towards treatments and cures.

The spotlighted research was funded by grants from the Core Center of Excellence in Hematology, Seattle Translational Tumor Research (STTR), and the Rett Syndrome Research Trust.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Keith Loeb, Siobhan Pattwell, Soheil Meshinchi, and Antonio Bedalov contributed to this study.

Loeb, A., Pattwell, S., Meshinchi, S., Bedalov, A., & Loeb, K. R. (2023). Donor Bone Marrow Derived Macrophage Engraftment into the Central Nervous System of Allogeneic Transplant Patients. Blood Advances, bloodadvances.2023010409. https://doi.org/10.1182/bloodadvances.2023010409