Cellular trafficking and fate mapping of cells within the nervous system after in utero hematopoietic cell transplantation

Data availability statement

All data regarding chimerism and CNS engraftment are retained within a database secured by the Washington University in St. Louis School of Medicine. Numerical source data for graphs and figures within the manuscript can be found in Supplemental Data 1 and 2 files. All histologic images, in addition to others not within the manuscript, are available upon request.

Mice

All experimental mice are derived from breeding colonies maintained within our animal housing facility. BALB/cJ and C57BL/6-Tg(UBC-GFP)30Scha/J (B6-GFP) colonies both originated from Jackson Laboratory (Bar Harbor, ME). Approval for the use of these mice was obtained from the Institutional Animal Care and Use Committee (Institutional protocols 20170217 and 20170236).

Injection technique

For all experiments, bone marrow is transplanted prior to birth in a competitive system without conditioning. In preparation for IUHCT, male BALB/cJ mice were singly housed and paired with female BALB/cJ mice for a 24-hour period in order to achieve time-dated mating. Donor bone marrow (BM) was harvested from the humeri, femora, and tibiae of adult B6-GFP mice of both sexes, unless otherwise noted. The presence of GFP allows for post-injection cell tracking. The cellular layers are separated using Ficoll Paque (GE Healthcare BioSciences, Marlborough, MA), then washed twice with phosphate buffered saline (PBS) to obtain bone marrow mononuclear cells (BM-MNC). Harvest and injection take place on the same day.

At gestational time points between 12 and 17 days (E12–E17), dams found to be pregnant on examination underwent anesthetic induction with isoflurane, then subsequent maternal laparotomy, followed by exteriorization of the uterine horns and a one-time intravenous (IV) injection of GFP⁺ donor BM-MNC into the vitelline vein (Supplementary Fig. 1). Injections are performed with a pulled glass needle of ~80–90 microns in diameter, using a Narishige IM300 nitrogen-driven micro-injector (Amityville, NY) under visualization with a dissecting microscope. To maximize potential engraftment, 20 × 10⁶ donor cells are administered to each fetus in 20 µL of PBS, which has been shown to be a safe and feasible volume⁷. The fetuses are then returned to the maternal abdomen until delivery at term, after which pups are placed with a foster mother (BALB-cJ mice) within 24 h of delivery to prevent transfer of donor-specific maternal antibodies through breastmilk⁵⁰.

Chimerism

Chimerism was defined as the percentage of cells that were CD45⁺/GFP⁺(Supplementary Data 2) To determine this percentage, flow cytometry was used either monthly or at time of terminal harvest. For monthly chimerism checks, mice underwent submandibular “cheek” bleeds. Blood was collected in 3 mL of PBS (ThermoFisher, Waltham, MA, USA) + EDTA (Invitrogen, Waltham, MA, USA). Samples were centrifuged for 5 min, and the supernatant discarded. Remaining plasma and red blood cells (RBC) were lysed for 5 min with RBC lysis buffer (G-Biosciences, St. Louis, MO, USA). The lysis reaction was stopped by the addition of ice-cold PBS + EDTA. Samples were centrifuged again for 5 min. The pellet was re-suspended in 100 µL of FACS staining buffer (Rockland Immunochemicals, Limerick, PA, USA) and stained with APC rat anti-mouse CD45 (BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min. Cells were washed with PBS, centrifuged for 5 min and resuspended in 500 µL of PBS. Samples were analyzed in conjunction with unstained BALB/cJ, CD45 APC stained BALB/cJ, and B6-GFP control specimens on a FACSCalibur3 (Becton-Dickson, Franklin Lakes, NJ, USA). Using FlowJo software (FlowJo, LLC, Ashland, OR, USA), gating was performed around leukocytes, and the percentage of cells that were both CD45 and GFP positive was used to determine the percentage chimerism (Supplementary Fig. 2).

Tissue sectioning

When treated animals reached the appropriate timepoints for harvest, they were terminally harvested. Mice were anesthetized with isoflurane, then cardiac puncture was done after sternotomy in order to collect peripheral blood samples. Mice were then perfused with 10–20 mL of PBS until the liver paled in color. The brain was then dissected out and placed in 4% para-formaldehyde (PFA) fixative (Sigma-Aldrich, St. Louis, MO, USA) in PBS, which was made prior to harvest. After 24–36 h of fixation, the specimens were cryoprotected in 30% sucrose in Tris-buffered saline (TBS) with 0.05% sodium azide (Sigma–Aldrich) for 48 h. Specimens were sectioned once the sample was no longer buoyant in the sucrose solution. The brains were divided at the cerebellopontine angle to separate forebrain from hindbrain (Supplementary Fig. 3). They were then serially sectioned in the coronal plane at 40 µm on a Microm HM430 freezing microtome (Microm International, Germany) and placed in an antifreeze cryoprotectant solution (Tris-buffered saline/30% ethylene glycol/15% sucrose/0.05% sodium azide) solution for long-term storage prior to their respective staining.

Tissue staining

Initial immunohistochemical staining against GFP was completed according to previously described protocols⁵¹. Endogenous peroxidase was inhibited by blocking sections with 1% H₂O₂ in TBS for 30 min. Sections were then rinsed three times with TBS, after which they were blocked with 15% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA, USA). Sections were then rinsed again with TBS three times and subsequentially incubated with rabbit anti-mouse GFP at 1:10,000 in a 10% NGS in 2% TBS-T solution for 2 h. Before proceeding, sections were rinsed in TBS again. They were then incubated for 2 h in a 1:1000 solution of avidin-biotin complex kit (ABC, Vector Laboratories, Burlingame, CA). Prior to the last staining step, sections were rinsed in TBS again. They were then incubated in 3,3’-diaminobenzidine (DAB) solution at 0.05% concentration with 0.01% H₂O₂ for roughly 10 min⁵². The DAB reaction was stopped with the addition of ice-cold TBS, after which the sections were rinsed and mounted onto chrome-gelatine-coated Superfrost Plus slides (VWR, Poole, UK) and air dried overnight. They were then dehydrated in ascending concentrations of ethanol (70–100%), cleared with xylene before finally cover-slipping with DPX mounting media (VWR). All tissue was stained with Balb/cJ as the negative control and B6-GFP as the positive control.

For immunofluorescent staining, tissue staining was completed as has been described by Nelvagal et al.⁵³. In brief, sections were slide mounted and air dried on SuperFrost Plus slides (Fisher Scientific, Waltham, MA, USA) for 30 min. They were then blocked with 15% normal goat serum (NGS) in 4% TBS-T (pH = 7.6). They were then incubated in the primary antibodies (rabbit anti-mouse GFP antibody, ab290, abcam®, Waltham, MA, USA, Table 3) at 1:1000 in 10% NGS in 4% TBS-T for 2 h, washed three times with TBS, at which point they underwent incubation in appropriate secondary antibody (Alexa Fluor^TM Goat anti-Rat 488 and 546, goat anti-rabbit 546 or goat anti-human 546, Invitrogen) at 1:200 concentration. The secondary antibody channel was chosen in 546 nm to eliminate the possibility of inherent GFP fluorescence confounding staining results. Slides were washed again three times with TBS, then treated with TrueBlack 1:20 in 70% ethanol (Biotium, Fremont, CA) to eliminate any background and/or autofluorescence. Slides were rinsed with TBS three times and coverslipped with DAPI Fluoromount G (Southern Biotech, Birmingham, AL, USA).

In situ hybridization (ISH) was completed with the use of RNAScope® probes and manuals⁵⁴. All reagents were RNAScope-specific unless mentioned otherwise. A female mouse who received an all-male donor transplant was terminally harvested at 3 months. Harvest took place according to previously described technique, except that the mouse was perfused with PBS alone, rather than PBS + EDTA. Brain specimen was fixed in PFA overnight, then placed in increasing concentration sucrose solutions of 10, 20, and 30%. Solutions were exchanged when the specimen was no longer buoyant. After equilibration within the 30% sucrose solution, it was frozen in O.C.T. Compound (Sakura, Torrance, CA, USA) on dry ice before being placed in a −80 °C freezer for storage. After equilibration at −20 °C for 1 h, it was then sectioned to 6 µm with sections mounted on SuperFrost Plus slides (Fisher Scientific) and air dried at −20 °C for 1–2 h. Slides were washed with PBS, then placed in a 60 °C oven for 30 min before cooling to room temperature. Slides were fixed again with 4% PFA for 15 min before proceeding to dehydration with 50% and 70% ethanol for 5 min each, with two final rinses in 100% ethanol for 5 min each. Slides were then air dried for 5 min before being placed in a 40 °C oven for 30 min. H₂O₂ was added at room temperature for 10 min before washing with deionized water twice. RNAScope® Retrieval Reagent was added to the slides while in a steamer for 5 min before washing in DI water. Slides were then washed in 100% ethanol for 3 min and dried at 60 C for 5 min. A hydrophobic barrier was made around the sections before Protease III was added with incubation for 30 min at 40 °C. Slides were washed with DI water before addition of a premade, pre-warmed probe mixture (40 °C for 10 min before cooling to room temperature). Incubation with RNAScope® probes (Advanced Cell Diagnostics, Newark, CA, USA) for GFP, Sex-determining region Y (SRY), and RNA Binding Fox-1 Homolog 3 (RBFOX3), took place at 40 °C for 2 h. Slides were rinsed in buffer for 4 min before incubation with AMP1 at 40 °C for 30 min. The same process was repeated for AMP2 and again for AMP3 but incubated for only 15 min. The HRP-C1 channel was developed for 15 min at 40 °C before washing with buffer, then adding secondary antibody. This process was repeated for HRP-C2 and HRP-C3, with all secondary concentrations at 1:1500 in Fluoroscein, Cy3.5 and Cy5.5 channels. DAPI was added at room temperature for 30 s before removal, then coverslipping with ProLong Gold Antifade mountant media (ThermoFisher).

For enteric staining proximal small bowel, distal small bowel, and colon/rectum were harvested in 6 cm segments. The bowel was then split along the mesenteric border and splayed into a planar fashion in 2 cm segments using pins and Sylgard 184 (Sigma–Aldrich®, St. Louis, MO, USA) covered petri dishes (Supplementary Fig. 4). The tissue was then fixed with 4% PFA before the muscularis and mucosa were separated under a dissecting microscope²². Muscularis and mucosa sections of duodenum, ileum, and colon were stained using a free-floating immunofluorescence protocol⁵⁵ for HuC/HuD, Glial Fibrillary Acidic Protein (GFAP), and GFP (Table 3) in order to evaluate neurons, glia, and donor-derived cells, respectively.

Microscopy

Stereomicroscopy was performed with a Leica M205FA stereoscope equipped with DFC7000T digital color camera. All image collection was performed using Leica LAS X software (Leica Microsystems, Buffalo Grove, IL, USA).

Widefield and fluorescence microscopy was performed with Zeiss Axioskop 2 MOT (Zeiss, Germany) with either a Zeiss Axiocam 506 color (Zeiss) or Hamamatsu ORCA-Flash fluorescence camera (Hamamatsu Photonics, Japan). Images were collected and analyzed with Stereo Investigator (MBF Bioscience, Williston, VT) and ImageJ (National Institutes of Health, Bethesda, MD, USA).

RNAScope-stained slides were imaged with Zeiss LSM 880 at 40X with images processed in Zen Lite (ZEISS Microscopy, Jena, Germany).

Analysis

For each gestational time point (E12-E17), mice were harvested at 1, 3, 6, 9, and 12 months of age in order to have a roughly equivalent number of mice in each group. Survival in E12 and E17 was limited, thus those groups are smaller in sample size. Working in batches, ~12 brains were stained at one time, then analyzed before proceeding to the next batch. This allowed for consistent stain intensity. Brain samples were separated into forebrain and hindbrain sections with every 36th section of forebrain and every 24th section of hindbrain analyzed.

Sections were then analyzed utilizing the software Stereo Investigator® (MBF Bioscience), and GFP⁺ cells were counted (Supplementary Data 1). Cells were excluded if they were associated with meninges, ventricles, or blood vessels in order to identify cells that were truly associated with the CNS. Each analyzed section was outlined in Stereo Investigator and volumetric analysis was performed using a Cavalieri volume estimator to standardize GFP cell counts to volume^27,52.

Volumetric analysis

Using Stereo Investigator (MBF Bioscience), sections were outlined, and their respective intervals documented in the software. All sections were 40 µm, allowing for volumetric analysis. A Cavalieri estimator was utilized with a 200 µm grid spacing for all samples. The total number of GFP⁺ cells, in addition to the volume estimate for over-correction, was entered into an Excel spreadsheet (Microsoft, Redmond, WA, USA). Given that the sections were only a sampling of the total specimen, the GFP⁺ cell count and volume were utilized in the below formulae to project an overall GFP⁺ cell concentration to volume, with a correction to mm³. Assuming that the evaluated sections encompass the anterior and posterior most portions of the brain, the section evaluation interval minus 1 was multiplied by the number of sections evaluated minus 1 in order to account for the first and last sections. This generated an assumed total number of sections which was then multiplied by the number of GFP⁺ cells yielded during manual counting. This result was the total number of GFP⁺ cells within the specimen. The Cavalieri volume estimator generated a volume corrected for OverProjection in order to account for errors in section outlining. This volume, in µm³, was converted to mm³ to bring numbers into a more easily manageable order of magnitude.

Statistical analysis

All data were statistically analyzed using GraphPad Prism for MacOS (GraphPad, San Diego, CA, USA). GFP⁺ cell concentrations were compared among gestational ages and among age at harvest using an unpaired, non-parametric ANOVA with Kruskal-Wallis multiple comparisons test. Forebrain and hindbrain concentrations were compared in a pooled fashion using paired parametric two-tailed t-test. Chimerism numbers were compared among gestational ages using a one-way ANOVA in an unpaired fashion. Chimerism numbers over time (months) were analyzed with a one-way ANOVA with linear trend test. Statistical significance was reached with p-value ≤ 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s42003-024-06847-6