Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

doi:10.21203/rs.3.rs-3757024/v1

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Research Article

Fetal liver CD34⁺ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

https://doi.org/10.21203/rs.3.rs-3757024/v1

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published 08 Jun, 2024

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Background

Transplantation of CD34⁺ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34⁺ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34⁺ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34⁺ isolated from cord blood (CB-CD34⁺) or fetal liver (FL-CD34⁺) and their engraftment into immunocompromised NOD/Shi-scid Il2rg^null (NOG) mice.

Methods

Three separate cohorts of NOG mice were transplanted with either CB-CD34⁺, FL-CD34⁺ or FL-CD34⁺ depleted of CD14⁺ endothelial cells to generate CB-NOG, FL-NOG and FL-CD14⁻-NOG, respectively. After 15–20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood, bone marrow, spleen, lungs and liver. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles.

Results

We show that CB-CD34⁺ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34⁺. In addition to HSPC, FL-CD34⁺ isolates contain non-hematopoietic CD14⁺ endothelial cells that enhance the engraftment of the human immune system in FL-CD34⁺-transplanted NOG (FL-NOG) mice. We demonstrate that these CD14⁺CD34⁺ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14⁺CD34⁺ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection.

Conclusion

Our results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

humanized mice

HSPC

fetal liver CD34

cord blood CD34

allogeneic tumor rejection

graft-versus-tumor

LSEC

sinusoidal dilatation

endothelial disfunction

Humanized immune system (HIS) mouse models, which are immunodeficient mice engrafted with human hematopoietic stem and progenitor cells (HSPC) and/or tissues, are utilized to study various human immune cells and predict human response to drugs (1, 2). NOD/Shi-scid Il2rg^null (NOG) (3) and NOD/LtSz-scid Il2rg^null (NSG) (4, 5) are the most common immunodeficient strains used for humanization. Both bare the severe combined immunodeficiency mutation Prkdc^scid on a nonobese diabetic (NOD) background and a mutation in the interleukin-2 (IL-2) receptor common gamma chain (5, 6). Different methods have been employed for the generation of HIS-mice over the years, using different sources of human cells and tissues and various routes of injection (1, 2). When transplanting exclusively HSPC, fetal liver (FL)-derived CD34⁺ (FL-CD34+) and cord blood (CB)-derived CD34⁺ (CB-CD34⁺) cells are injected intra-hepatically or via other routes into new-born mice (5, 7–14) or via the tail vein in adult mice (3, 7, 8, 15–22). A single injection of human HSPC is technically simple and medium (10–20 mice) to large (> 30) cohorts of HIS mice can be generated from a single donor of FL or CB, respectively (23). These HIS mice are generally healthy or develop GVHD after long-term engraftment, which allows a larger window for experimentation (7, 12, 24, 25).

FL is an attractive source of CD34⁺ HSPC, since > 1 x 10⁸ cells can be isolated from a single donor (26). This enables the generation of large and/or multiple cohorts of HLA-defined humanized mice for tumor studies. When humanized mice are reconstituted with FL-CD34⁺ and used for cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) engraftment, partial HLA matching is routinely applied (17, 27, 28). The latter is to prevent tumor rejection by alloreactive T cells, also known as graft versus tumor (GVT) effect (29, 30). When CB-CD34⁺ are used for reconstitution, HLA matching is not required for successful tumor growth and the HLA alleles are usually not mentioned or found irrelevant (18, 21, 22, 31–36). This discrepancy suggests functional differences between FL- and CB-CD34⁺-reconstituted mice that might arise from distinct phenotypical characteristics. Importantly, as various research groups use different methods for mouse humanization in terms of CD34⁺ isolation, conditioning protocols, strain and age of mice, injection site and numbers of injected cells, a direct comparison of different CD34⁺ sources for humanization has yet to be accomplished.

Here, we have isolated CD34⁺ cells from CB and FL samples and found significant differences in their phenotype and reconstitution potential in adult NOG mice, referred as CB-NOG and FL-NOG, respectively. Our results reveal that FL-CD34⁺ isolates contain a population of CD14⁺ endothelial cells that enhances the reconstitution of human immune cells in FL-NOG.

These FL-CD34⁺CD14⁺ cells support the engraftment of human liver sinusoidal endothelial cells (LSECs) in FL-NOG but contribute to hepatic sinusoidal dilatation which can ultimately progress to a severe pathology that resembles graft-versus-host disease (GVHD). Finally, by employing an HLA-mismatched melanoma model, we show that graft-versus-tumor (GVT) can be induced in FL-NOG, but not CB-NOG. Therefore, the FL-NOG mice might serve as a novel model to study human LSEC disfunction and mechanisms of solid tumor rejection.

Mice

NOD/Shi-scid Il2rg^null (NOG) (3) and NOG-HLA-A24 transgenic (Tg) mice were obtained from The Central Institute for Experimental Animals (CIEA, Japan). NOG-HLA-A24 Tg mice were established by backcrossing HLA-A24 Tg (C57/BL6) from Taconic Biosciences (Albany, NY, USA) onto the NOG background, as described previously for NOG-HLA-A2 Tg (37). Mice were shipped from CIEA, Japan at 5–6 weeks of age to the Biological Resource Centre (BRC, A*STAR, Singapore) and let to acclimatize for a minimum of 7 days before any procedures were performed. Animals were housed in the BRC facility under specific pathogen-free conditions, on controlled 12h light/dark cycles, and food and water ad libitum. Enrichment domes and nesting sheets were placed in all the cages.

Isolation of Human HSCs

Fetal liver tissue was aseptically collected, transported on ice and immediately immersed in cold phosphate buffered saline (PBS) with amphotericin B (5 µg/ml) (Fungizone), penicillin (200 U/ml) and streptomycin (200 µg/ml) (all from Gibco) for 10 minutes at 4°C. A cell scraper was used to gently dissociate the tissue into a collagenase/dispase digestion medium (Liver Digest Media, Gibco) supplemented with 4U/ml DNase I (Invitrogen). The tissue was digested for 20 minutes at 37°C, homogenized, filtered and the obtained suspension overlayed on Ficoll-paque Plus (Cytiva, MA, USA). Cord blood was diluted in a 1: 1 ratio with PBS and overlayed on Ficoll-paque Plus for density cell centrifugation. CD34⁺ HSPC were enriched from the mononuclear layer by two rounds of magnetic positive selection using the Ultrapure Human CD34 MicroBead Kit (Miltenyi Biotec, Germany). Purity of CD34⁺ HSPC cells was assessed by flow cytometry (Additional file 1: Fig. S1 and Additional File 2: Table S1), and cells were cryopreserved in Bambanker (GC Lymphotec, Japan).

FL-CD34⁺ sorting and cytospin

FL-CD34⁺ were thawed, stained for Fluorescently Activated Cell Sorting and sorted on FACSAria (BD Biosciences) to deplete CD14⁺ cells as shown in Additional file 1: Fig. S2. For morphology assessment, the sorted CD14⁺ and CD14⁻ fractions were cytospined onto slides using a Shandon Cytospin 4 centrifuge (Thermo Scientific). Eosin and haematoxylin-stained slides were mounted with Entellan rapid mounting medium (Merck), imaged at 100x magnification using light microscope (Olympus BX43) and photomicrographs captured by an Olympus DP21 camera.

Human cell transplantation

6 weeks old male NOG mice were sub-lethally irradiated with 1.2Gy (RS 2000 X-Ray irradiator; Rad Source Technologies Inc., GA, USA) and injected with 5 × 10⁴ live human CB-CD34⁺, FL-CD34⁺ or an equivalent number of live FL-CD34⁺CD14⁻ in PBS through the tail vein (i.v.). Three independent cohorts of mice were generated, using three different CB and FL donors, with no specific randomisation method for mouse or cage allocation. Detailed mouse information can be found in Additional File 2, Table S2. Mice were monitored weekly for any adverse effects, including weight loss, pallor, hair loss and weakness. As the experiment was designed to directly compare two groups, no exclusion criteria were set a priory, however three experimental animals were excluded due to unexpected death (two accidental and one unspecified; details in Additional File 2, Table S2).

For CDX experiments, 6–8 weeks old male and female NOG and NOG-HLA-A24 Tg mice were sub-lethally irradiated with 1.2Gy and injected i.v. with 5 x 10⁴ CB-CD34⁺ or 7.5 x 10⁴ FL-CD34⁺ in PBS. Four independent cohorts of mice were reconstituted with one CB-CD34⁺ and two FL-CD34⁺ donors, with no specific randomisation method for mouse or cage allocation. Detailed mouse records are found in Additional File 2, Table S3.

The various group allocation was known to everyone at different stages of the experiment, except to the pathologist to whom the groups were revealed only after the pathology assessment and scoring.

Cell line-derived xenograft (CDX) tumor model

After confirming the reconstitution of T cells (14–16 weeks post HSPC transplantation), the humanized mice were first injected intraperitoneally (i.p.) with 3 doses of 10 µg Flt3L-Fc (BioXcell) given every 2 days to induce the generation of DCs (38). This was followed by i.p. immunization with 1 x 10⁶ non-viable A375 melanoma tumor cells treated with mitomycin-C (Sigma-Aldrich). After 7 days of priming, mice were subcutaneously injected with 1 x 10⁶ viable A375 cells in the right flank and tumor growth was measured twice a week using calipers (schematic of the experiment in Addional File 1, Figure S3). Animals were monitored bi-weekly for tumor necrosis and adverse effects such anemia or skin GVHD (pallor, weight loss, weakness, hair loss) and euthanized on humane grounds in case of necrotic tumor or severe symptoms. As all the outcomes were important for the study, including the development of any adverse reactions, no experimental animals were excluded from the study. For records of tumor rejection and adverse reaction frequency see Additional File 2, Table S3.

Humanized mouse tissue isolation and processing

Graduated capillaries with heparin were used to collect 100µl peripheral blood from the retro-orbital venous plexus of isoflurane-anesthetized mice at 4, 8, 12, 15 and 20 weeks post-CD34 + transplantation. Red blood cells were lysed with Pharm Lyse (BD Biosciences). For endpoint analyses mice were sacrificed by CO₂ inhalation at 15 or 20 weeks post transplantation. Bone marrow (BM) samples were collected by flushing one femur with 1ml of cold PBS. Spleens were crushed with a syringe plunger, digested with 0.2 mg/ml collagenase IV (Sigma-Aldrich, MO, USA) supplemented with 0.03 mg/ml DNAse I (Roche Diagnostics GmbH, Mannheim, Germany) for 30min at 37°C. Lungs were cut into smaller pieces with scissors, digested with 1.4 mg/mL Collagenase A (Merck) and 0.03 mg/ml DNase I for 1h at 37°C. Livers were cut into smaller pieces and digested with 0.2 mg/ml collagenase IV with 0.05 mg/ml DNAse for 45min at 37°C. Liver single cell suspensions were resuspended in 40% isotonic Percoll PLUS (Sigma-Aldrich) and centrifuged to remove the top layer of fat and tissue cells. ACK lysis buffer (Gibco) was used to lyse red blood cells.

Flow Cytometric analysis of blood and tissue-infiltrating immune cells

For reconstitution kinetics, blood cells were stained with the antibodies listed in Additional file 2: Table S4, acquired on FACSCanto II (BD Biosciences) and gated as shown in Additional file 1: Fig. S4. Antibody panels for endpoint analyses of blood, bone marrow, spleen and lungs can be found in Additional file 2: Table S5, with gating strategies in Additional file 1: Fig. S5. Non-specific staining was blocked with human (TruStain FcX™, BioLegend) and mouse (2.4G2 hybridoma supernatant) Fc block for 15 min. Extracellular markers were stained at 4°C for 30min. For intracellular staining, BM, spleen and liver samples were permeabilized and fixed using the BD Cytofix/Cytoperm kit following the manufacturer’s instruction. Lung cells were fixed in 2% PFA. Live/dead exclusion was achieved by resuspending in DAPI (Invitrogen) for unfixed blood cells, while all fixed samples were pre-stained with the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen). The samples were acquired on a LSR5 (BD Biosciences) with FACSDivaTM software (BD Biosciences). Analysis was performed using Flowjo Software (v. 10.4.0) (Flowjo, LLC).

Histology

Tissues were harvested and fixed in 10% NBF for about 48h at RT. Fixed tissues were processed in ascending grades of alcohol, cleared in Xylene, infiltrated with paraffin wax and cut at 5µm thickness. The tissue sections were brought to water and stained with Haematoxylin and Eosin (H&E). Histo-pathological assessment was performed by pathologist in a blind fashion using Olympus BX51 upright microscope and representative photomicrographs were captured with an Olympus DP71 digital colour camera and cellSens Imaging software (Olympus Life Science, Japan).

Multiplex Microbead-Based Immunoassay

MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel – Premixed 41 Plex- Immunology Multiplex Assay (Merck/Millipore) was used to measure serum cytokines. Samples were acquired on the FLEXMAP® 3D (Luminex) using xPONENT® 4.0 (Luminex) software and data analysed with Bio-Plex ManagerTM 6.1.1 (Bio-Rad).

ELISAs

ALT was measured in plasma with the Mouse ALT Elisa Kit (Wuhan Fine Biotech Co., Ltd) according to the manufacturer’s instructions. Factor VIII was measured in the plasma with the Human FVIII ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's instructions, where human plasma was diluted 1:50 and humanized mouse plasma 1:2.

HLA Typing

DNA was extracted from the CD34⁻ fraction of CB and FL samples using the PureLink® Genomic DNA Kit (Invitrogen). Sequence based typing (HLA Laboratory, Blood Services Group, Health Sciences Authority, Singapore) was used to determine the genotype of the HLA-A alleles.

Statistical methods

GraphPad Prism 9 (GraphPad Software) was used to prepare graphs and perform statistical comparisons. The Kolmogorov-Smirnov test was used for normality calculations. Two groups without normal distribution were compared with the Mann-Whitney test and, where indicated, corrected for multiple comparisons using the Holm-Sidak method. Three groups were compared with an ordinary one-way ANOVA with post-hoc Tukey if normally distributed or alternatively, with a Kruskal-Wallis test with post-hoc Dunn’s. All correlations were calculated using Spearman correlation, as the pathology scoring data was not normally distributed. P values < 0.05 were considered statistically significant.

CD34⁺ cells isolated from fetal liver have a distinct phenotype compared to cord blood- CD34⁺

CD34⁺ cells isolated from CB and FL were evaluated for purity and phenotypical characteristics. The frequency of CD34⁺ from both sources was similar (> 95%) (Additional File 1: Fig S1A,B), however a considerable proportion of FL-CD34⁺ was positive for lineage (lin) markers CD3/CD14/CD16/CD19/CD20/CD56 (Fig. 1A,B, Additional File 1: Fig S1A). This lowered the fraction of CD34⁺lin⁻ in FL-CD34⁺ to 66.5% (Additional File 1: Fig. S1C) and prompted us to perform in-depth phenotyping of the CD34⁺ isolates. By using an extended flow cytometric panel (Additional File 2: Table S6), we found that FL-CD34⁺ consist of at least three distinct populations: lin⁻ (indigo), lin⁺CD38^high (orange) and CD14⁺ (magenta) (Fig. 1A-C). Contrary, CB-CD34⁺ demonstrated a relatively uniform population of CD34⁺lin⁻CD14⁻ (lin⁻) cells (Fig. 1A-C). Based on lower expression of CD45 and the presence of endothelial markers CD10 and CD90 (Fig. 1C,D and Additional File 1: Fig. S6A), we classified the CD14⁺ cells as fetal endothelial cells, possibly liver sinusoidal endothelial cells (LSECs) (39). We confirmed the endothelial morphology by Giemsa staining, showing a characteristic vacuolated and blebbing cytoplasm (40, 41) (Fig. 1E, left panel). In contrast, the CD34⁺CD14⁻ fraction presented with a high nuclei-to-cytoplasm ratio, typical of blast/stem cells (41) (Fig. 1E, right panel).

Lastly, we compared the progenitor populations contained within the CD34⁺lin⁻CD14⁻ (42). By applying a Fluorescence Minus One (FMO) control for CD38, we determined that the majority of CD34⁺ isolated from both sources are CD38⁺ late progenitors (Additional File 1: Fig. S6B). Overall, the progenitor profile of CB- and FL-CD34⁺lin⁻CD14⁻ cells was similar, except for frequencies of common dendritic cell (DC) progenitors (CDP) and monocyte-DC progenitors (MDP), which were significantly higher among FL-CD34⁺lin⁻CD14⁻ (Fig. 1F,G). In summary, CB-CD34⁺ consist predominately of lin⁻ HSPC, while FL-CD34⁺ cells additionally contain lineage-committed lin⁺CD38^high progenitors and non-hematopoietic CD14⁺ endothelial cells.

FL-NOG engraft human endothelial and immune cells

As the composition of FL-CD34⁺ significantly differs from CB-CD34⁺ (Fig. 1), we predict that transplanting equal numbers of FL-CD34⁺ and CB-CD34⁺ in NOG might lead to differential reconstitution of immune cells. To evaluate this, we performed a comprehensive side-by-side longitudinal analysis of CB-CD34⁺ and FL-CD34⁺-transplanted NOG mice (CB-NOG and FL-NOG; respectively) (Fig. 2A). The CD14⁺ endothelial cell population contained within FL-CD34⁺ might affect the reconstitution of the hematopoietic stem cells (10, 43) and/or contain engraftable CD14⁺CD34⁺ LSECs or their precursors (39, 44). Hence, we also transplanted NOG with CD14⁺-depleted FL-CD34⁺ (FL-CD14⁻-NOG) (Additional File 1: Fig. S2) alongside total FL-CD34⁺.

The kinetics of human immune CD45 (hCD45) cell reconstitution in the blood of CB-NOG was faster than in FL-NOG by percentages and absolute numbers (Fig. 2B,C). CD14⁺ depletion additionally delayed hCD45 reconstitution in FL-CD14⁻-NOG (Fig. 2C). CB-NOG displayed increased reconstitution of hCD19⁺ B cells compared to FL-NOG within the first 12 weeks post-transplant (wpt; Fig. 2D,E). Similarly, CD3⁺ T cell numbers were higher in CB-NOG at 12 and 15 wpt (Fig. 2F,G). On the other hand, FL-NOG reconstituted higher frequencies of CD33⁺ myeloid cells at 8, 12 and 15 wpt (Fig. 2H). Absolute numbers of CD33⁺ and CD19⁺, but not CD3⁺, were lower at 12 wpt in FL-CD14⁻-NOG in comparison to FL-NOG (Fig. 2E,G,I). Overall, transplantation with FL-CD34⁺ results in a slower reconstitution of the human immune system compared to CB-CD34⁺, which is further delayed by the depletion of CD14⁺ endothelial cells. However, FL-NOG reconstitute a larger proportion of CD33⁺ myeloid cells, in accordance with a higher frequency of MDP and CDP within FL-CD34⁺ (Fig. 1F,G).

LSECs are among the main producers of Factor VIII (FVIII) (45) and human FVIII can be detected upon successful LSEC engraftment in humanized mice (39, 44). Thus, to evaluate human endothelial cell engraftment, we measured human FVIII in the plasma of humanized NOG and compared it to human samples. Our data clearly demonstrates that the engraftable FVIII-producing LSECs are contained within FL-CD34⁺ and are completely removed by CD14⁺-depletion (Fig. 2J).

Differences in long-term multi-lineage reconstitution of the human immune system in CB- and FL-NOG mice

Upon assessing the chimerism in the peripheral blood, we next evaluated the differential multilineage reconstitution of human immune cells in various tissues at endpoint (Additional File 2: Table S5, gating strategies in Additional File 1: Fig. S5). At 20 wpt the frequency of T cells, specifically CD4⁺ T, continued to be higher in the blood of CB-NOG compared to FL-NOG (Fig. 3A). The frequencies and numbers of all other analysed immune cells were comparable (Fig. 3A and Additional File 1: Fig. S7A). In the bone marrow (BM) we detected human neutrophils at various stages of development (Additional File 1: Fig. S4B) and the proportion of mature CD66b⁺CD16^high neutrophils was significantly higher in FL-NOG compared to CB-NOG (Additional File 1: Fig. S7B). Except for two mice, no mature human neutrophils were found in the peripheral blood of humanized NOG mice irrespective of the CD34⁺ source (Fig. 3A and Additional File 1: Fig. S7A). Though, almost all animals had detectable neutrophils in the spleen, lung and liver and FL-NOG had significantly more neutrophils within all the analysed organs (Fig. 3B-F and Additional File 1: Fig. S7C,D). Additionally, FL-NOG spleens contained more HLA-DR⁺CD68⁺ macrophages (Fig. 3B), and FL-NOG livers had more abundant CD11c⁺ dendritic cells (DC) and auto-fluorescent macrophages (Fig. 3E). Several myeloid populations (cDC1, CD163⁺ and CD163⁻ macrophages) were also more frequent in the lungs of FL-NOG (Fig. 3C).

Depleting CD14⁺ cells prior FL-CD34⁺ transplantation significantly decreased the numbers of human CD4⁺T, CD8⁺T, NK cells, neutrophils and macrophages within the liver (Fig. 3G). We observed a strong linear correlation between blood and liver hCD45 chimerism in FL- and FL-CD14⁻-NOG (Fig. 3H). However, total liver hCD45⁺ numbers did not increase along with blood chimerism in FL-CD14⁻-NOG mice (Fig. 3I), suggesting endothelial CD14⁺ cells might be important for immune cell recruitment or homing within the liver. To summarize, the increased reconstitution of several myeloid populations in FL-NOG compared to CB-NOG was consistent with an increased frequency of myeloid progenitors contained within FL-CD34⁺. Additionally, our data suggests that the endothelial FL-CD34⁺CD14⁺ may support the recruitment of FL-CD34⁺CD14⁻ reconstituted immune cells to the liver.

FL-CD34⁺-transplanted NOG mice develop a distinct GVHD liver pathology

Long-term reconstitution of the human immune system in immunodeficient mice can lead to xenogeneic graft-versus-host-disease (GVHD) (9, 12, 46, 47). In our experiments all mice survived until endpoint without weight loss (not shown), however 3 out of 11 (27%) FL-NOG mice developed visible dark spots on the liver (Fig. 4A) and 2 out of 12 (16.7%) CB-NOG developed facial alopecia (Additional File 1: Fig. S8A). The cumulative liver pathology score was most severe in FL-NOG (Fig. 4B and Additional File 1: Fig. S8B). Histopathological analysis identified that the dark spotting of the liver was due to multifocal haemorrhages, which were associated with multifocal hepatic necrosis (Fig. 4C, Additional File 1: Fig. S8B). Sinusoidal dilatation (SD) was present in 10 out of 11 (91%) FL-NOG livers, even when no other pathology was present (Fig. 4D; Additional File 1: Fig. S8B,C). FL-CD14⁻-NOG had a significantly reduced incidence of these phenotypes (Fig. 4B,D), suggesting that human LSEC engraftment contributes to the liver pathology in FL-NOG.

To identify specific cell types and mediators associated with the overall liver pathological score, we performed Spearman correlation analyses (Additional File 2: Tables S7,8). Liver pathology in FL-NOG correlated positively with both lymphoid and myeloid cell numbers across multiple tissues (Fig. 4E). Whereas, in CB-NOG, it positively correlated only with human lymphoid cell numbers, specifically with CD4⁺ T, CD8⁺T and NK in spleen and/or liver (Fig. 4E). Multiple cytokines and chemokines were associated with the pathology score in both CB- and FL-NOG, including CCL2/MCP-1 (Fig. 4F, Additional File 1: Fig. S8D, Additional File 2: Table S8). Increased levels of alanine transaminase (ALT) positively correlated with liver inflammation exclusively in CB-NOG (Fig. 4G), while CXCL10 was among the cytokines important for driving the pathology solely in FL-NOG (Fig. 4H, Additional File 1: Fig. S8D, Additional File 2: Table S8). LSEC have been shown to produce CXCL10 and CCL2 under inflammatory conditions upon acute liver injury (48). Depletion of CD14⁺ LSEC-reconstituting cells prior transplantation of FL-CD34⁺, significantly decreased serum levels of human CXCL10 and CCL2 (Fig. 4I).

In summary, long-term reconstitution with CB- and FL-CD34⁺ can lead to distinct GVHD-associated pathological changes in NOG mice. In FL-NOG we detected a previously undescribed spotted liver pathology characterized by sinusoidal dilatation, systemic increase in lymphoid and myeloid cell numbers and secretion of CXCL10. This GVHD-like phenotype was found to be due to presence of endothelial CD14⁺ cells among FL-CD34⁺.

Allogeneic solid tumors are rejected in FL-CD34⁺-reconstituted mice

To assess the functionality of CB- and FL-CD34⁺ reconstituted mice, we next tested their ability to mount an allogeneic anti-tumor response using an A375 melanoma CDX model (Additional File 1: Fig. S3, HLA-A alleles in Additional File 2: Table S9). In conventional NOG mice, the human T cells are selected and educated in the mouse thymus on mouse MHC (49). To stimulate the development of human HLA-A*24 restricted T cells from A*24:02 donor HSPCs, we first opted for NOG mice that express human HLA-A*24:02 (NOG-HLA-A24 Tg or A24 Tg in short). Except for one mouse, A375 CDX were not rejected in mice reconstituted with HLA-A mismatched CB-CD34⁺ (Fig. 5A,B). In contrast, A375 CDX did not grow well in mice reconstituted with mismatched FL-CD34⁺ and after being initially palpable, tumors were completely rejected in > 50% of the mice (Fig. 5B). To test whether the HLA-A Tg was necessary for the observed rejection, we repeated the same experiment in conventional NOG mice transplanted with two FL donors, FL-4 and FL-13. In both cases, > 50% of mice completely rejected the tumors (Fig. 5C,D). In these experiments, we transplanted the mice with 7.5 x 10⁵ FL-CD34⁺ to achieve similar reconstitution levels to CB-CD34⁺ (Additional File 1: Fig. S9A). The higher cell dose resulted in a high incidence of the spotted liver phenotype, which was 50–100% depending on the cohort. Donor FL-4 induced a more severe pathology compared to FL-13 by visual inspection with corresponding higher levels of serum cytokine/chemokine (Fig. 5E, Additional File 1: Fig. S9B). Additionally, FL-4 reconstituted NOG mice that did not completely reject the tumors, were significantly more prone to develop weakness and pallor leading to premature death or requiring euthanasia (Additional File 1: Fig. S9C). Failure of complete rejection resulted in increased serum cytokine/chemokine levels (Fig. 5F), suggesting that the underlying GVHD was exacerbated by tumor burden. In summary, allogeneic GVT can be achieved in FL-NOG, however it comes at the expense of developing liver GVHD.

We present here a detailed comparison of CB-CD34⁺ and FL-CD34⁺-engrafted NOG mice, including characterization of CD34⁺ isolates, reconstitution kinetics, endpoint analyses of tissue immune cells, histopathological liver assessment and the ability to reject HLA-mismatched tumors. There are few reports that have compared CB- to FL-CD34⁺-reconstituted NSG mice with contradictory results. When equal numbers of CD34⁺ were injected, Patton et al. showed superior engraftment of FL-CD34⁺ vs CB-CD34⁺ (14), while the opposite was observed by Chiorazzi et al. (50). By using increasing numbers of FL-CD34⁺, another study reported a non-linear association with blood reconstitution, showing slow engraftment with < 4 x 10⁴ cells and rapid engraftment when 5 x 10⁵ cells were used (11). We similarly show that injecting 5 x 10⁴ FL-CD34⁺ resulted in slow reconstitution kinetics (Fig. 2C) which improved substantially when we increased the cell dose to 7.5 x 10⁴ (Additional File 1: Fig. S9A).

A major confounding factor that hinders the comparisons of CB- and FL-engrafted mice is the CD34⁺ isolation method. While the isolation from CB is straightforward, the FL requires a digestion step that varies from laboratory to laboratory in terms of collagenase, time and temperature used (11, 13, 14, 51–55). Additionally, some researchers use density gradient to enrich HSPCs (13, 14, 50, 53, 54), while others do not (11, 12, 51, 52). Some isolation techniques can enrich CD34^lo hepatic/mesodermal progenitors (10, 56) and whole liver extracts are known to contain non-hematopoietic CD34⁺ endothelial cells (55, 57). Using the isolation methods described here, we show that CD34⁺ cells isolated from CB represent a relatively uniform population, while FL-CD34⁺ are composed of several distinct populations (Fig. 1), in accordance with a previous report (56). Two recent studies have explored the transcriptome of fetal liver cells on a single-cell level (40, 58). The study by Popescu et al. identified three populations expressing CD34, that could be potentially isolated using anti-CD34 magnetic beads: (i) hematopoietic stem cells and multipotent progenitors (MSC/MPP), (ii) CD38^high pre-pro-B cells and (iii) CD14⁺ endothelial cells (40). These likely correspond to the (i) lin⁻ HSPC, (ii) lin⁺CD38^high and (iii) CD14⁺ endothelial populations we identified in our FL-CD34⁺ isolates. Within lin⁻ HSPC we detected higher myeloid progenitor frequencies in FL-CD34⁺ compared to CB-CD34⁺, which upon transplantation resulted in an increased proportion of CD33⁺ myeloid cells in the blood of FL-NOG. Additionally, FL-NOG mice consistently harboured higher numbers of CD16^hiCD66b^hi neutrophils across organs. This is likely due to increased development in the bone marrow and warrants further investigation in models that support better neutrophil reconstitution, such as hG-CSF KI NOG (59). FL-NOG spleens, lungs and liver also contained more macrophages and dendritic cells, possibly due to higher myeloid progenitor frequencies. However, the increased myeloid cell infiltration is more likely associated with the inflammatory environment that we uncovered in FL-NOG because of CD14⁺ endothelial cell transplantation (Fig. 4).

CD34⁺CD14⁺ endothelial cells represent approximately 2–4% of total digested fetal liver cells (40). CD14⁺ LSECs from fetal liver have been shown to engraft in urokinase-type plasminogen activator transgene (uPA-NOG) mice and produce FVIII (39). FVIII-producing LSECs and other non-parenchymal hepatic cells were also detected in MISTRG6 mice upon engraftment of FL-CD34⁺ (44). Here we show that LSECs can engraft in simple NOG mice and enhance the reconstitution of the human immune cells. Removing CD14⁺ cells from FL-CD34⁺ before transplantation significantly delayed the engraftment of the human immune system in NOG mice, specifically affecting CD19⁺ and CD33⁺ cell numbers (Fig. 2E,I). LSECs have been shown to promote HSPC expansion ex vivo and might support reconstitution in vivo through the secretion of yet to be defined factors (43). Another possibility is that human LSEC engraftment induces some sort of damage to the mouse sinusoidal endothelium. This could lead to immune activation and recruitment of immune cells into the liver and other organs through the secretion of mediators such as CCL2 and CXCL10, ultimately resulting in liver injury (48).

Long-term reconstitution of immune cells can cause spontaneous activation of the human immune system in humanized mice, resulting in different manifestations of xenogeneic GVHD. CD34⁺ isolated from CB and GM-CSF-mobilized peripheral blood have been shown to induce a form of skin GVHD (GVH-S) characterized by alopecia and scaly skin with or without weight loss (24, 25, 47, 60). GVH-S develops in a donor-specific manner and seems to be associated with certain HLA haplotypes (47, 60). Our study confirmed donor-dependent facial alopecia in CB-NOG associated with increased T and NK cell numbers in the spleen and liver. Another type of GVHD with multisystemic granulomatous has been identified in mice reconstituted with G-CSF-mobilized peripheral blood (MBP)-CD34⁺ (61, 62), CB-CD34⁺ (63) and FL-CD34⁺ (12). T cells and macrophages have been found in BM, liver and lung infiltrates of these animals (12, 62, 63). The liver pathology observed in FL-NOG from our study was similarly correlated with both CD4⁺ T cells and macrophages, specifically CD14^highCD68⁺ that correspond to the inflammatory CD32^mid Kupffer cells identified by Wu et al (64). The increased soluble factors, including CXCL10 and CCL2, likely contributed to the recruitment of T and NK cells and egress of myeloid cells from the BM resulting in increased organ infiltration (Fig. 6). We have uncovered that sinusoidal dilatation (SD) was the most distinctive feature present in FL-NOG livers. SD can be caused by endothelial dysfunction, which is a common complication associated with tissue rejection (65–67). Taken together, these findings suggest that the spotted liver phenotype is likely a form of GVHD (68, 69). Removing endothelial CD14⁺ cells from the FL-CD34⁺ preparations not only delayed blood reconstitution, but also significantly reduced serum CXCL10 and CCL2 and human cell infiltration into the livers of transplanted NOG mice. Consequently, none of the FL-CD14⁻-NOG developed the spotted liver phenotype.

Finally, we investigated the functionality of the human immune system in CB- and FL-NOG by engrafting an HLA-A-mismatched melanoma CDX. We observed complete tumor rejection in ≥ 50% of FL-NOG or FL-NOG-A24 Tg, but not in CB-NOG. The HLA-A24 Tg was not necessary for the tumor rejection and majority of the mice developed the spotted liver GVHD phenotype. These observations suggests that the graft-versus-tumor (GVT) effect is either due to a better functionality of the immune system, associated with the GVHD responses or a combination of both (70). The goal of allogeneic hematopoietic cell transplantation in a clinical setting is to achieve GVT without inducing significant GVHD (70). In our study, we observed a severe wasting GVHD leading to experimental animal loss, primarily in mice that failed to reject tumors. This suggests that the underlying GVHD can be intensified by tumor burden or possibly by hypercytokinemia induced by the ongoing tumor lysis (71). Due to the overlapping characteristics of these conditions, further studies are needed to elucidate the mechanisms involved in endothelial dysfunction, GVHD and GVT in FL-NOG mice.

Taken together our study highlights the importance of deeper phenotyping of FL-CD34⁺ isolates prior transplantation. As we have shown here, the FL contains multiple CD34⁺-expressing populations that can have a profound impact on the phenotypes of transplanted animals. Our study provides insights into a novel form of GVHD that develops in FL-NOG, which can be attributed to the CD14⁺ endothelial cells contained within FL-CD34⁺ isolates (Fig. 6). Finding strategies to prevent or delay the loss of immunological tolerance towards the host is crucial for the development of models requiring stable long-term engraftment. On the other hand, FL-NOG might serve as a unique model to study GVT responses and possible strategies to prevent the underlying endothelial dysfunction and GVHD.

ALT: alanine transaminase

BM: bone marrow

CB: cord blood

CB-NOG: NOG mice transplanted with CB-CD34⁺

CDP: common dendritic cell progenitors

CDX: cell line-derived xenograft

DC: dendritic cell

FL: fetal liver

FL-NOG: NOG transplanted with aFL-CD34⁺

FL-CD14^-- NOG: NOG transplanted with CD14⁺-depleted FL-CD34⁺

FMO: fluorescence minus one

FVIII: Factor VIII

GVHD: graft-versus-host disease

GVH-S: skin graft-versus-host

GVT: graft-versus-tumor

H&E: Haematoxylin and Eosin

HIS: Humanized immune system

HLA: human leukocyte antigen

HSPC: hematopoietic stem and progenitor cells

Lin: lineage

LSEC: liver sinusoidal endothelial cells

MDP: and monocyte-DC progenitors

NOG: NOD/Shi-scidIl2rg^null mice

NSG: NOD/LtSz-scidIl2rg^null mice

PDX: patient-derived xenograft

PBS: phosphate buffered saline

SD: sinusoidal dilatation

Tg: transgenic

uPA-NOG: NOG mice expressing transgenic urokinase-type plasminogen activator in the liver

WPT: weeks post-transplant

Ethics approval and consent to participate

All animal experiments were performed in accordance with approved protocols by the Biological Resource Centre (BRC, A*STAR, Singapore) Institutional Animal Care and Use Committee (IACUC) as follows: #161192 titled “Production of HLA matched humanized NOG mice” approved in January 2017, #19496 titled “Service Protocol for the Production of Humanized NOG mice” approved in June 2020 and #181406 titled “Study of human tumor-immune therapy by generating HLA matched NOG mice model with human tumor cell lines” approved in March 2019. The reporting of animal experiments adheres to the ARRIVE guidelines.

Human cord blood from full-term uncomplicated pregnancies and fetal liver tissue from clinically indicated termination of pregnancies of gestation age 14 to 21 weeks were collected at the KK Women’s and Children’s Hospital, Singapore. All the donations were performed in compliance with the SingHealth Centralised Institutional Review Board (CIRB) guidelines and written informed consent for tissue donation was obtained from all donors. CIRB #2013/778/D titled “Derivation of Stem Cells from Perinatal Birth Tissues” CIRB #2013/837/D titled “Ontogeny of the Fetal Immune System” were first approved in November 2013 and both protocols are renewed yearly. For human plasma sample collection, the study was approved by the National Healthcare Group (NHG) Domain Specific Review Board (#DSRB-2011/01917). Written informed consent was obtained from the participants before recruitment.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

The CIEA-KKH-SIgN Humanized NOG Mice for Immuno Therapy (HuNIT) platform is supported by the Industry Alignment Fund – Pre-positioning Programme (IAF-PP; #H16/01/a0/002). JKYC is supported by Singapore’s Ministry of Health (MOH-001221-01, MOH-000932-00) and National Medical Research Council (CIRG/1484/2018, NMRC CSA (SI)/008/2016, NMRC STaR22jul-0004). The Immunomonitoring platform is supported by the National Research Foundation (NRF), Immunomonitoring Service Platform (ISP) grant (Ref: NRF2017_SISFP09).

Authors' contributions

Contribution: T.C. designed and performed the experiments, analysed the data, and wrote the manuscript; L.X., Z.Y. and A.L. performed experiments and analysed data; T.H.C. performed experiments, L.H.H., E.K.H, F.Y., J.K.Y.C. and A.M. assisted with regulatory approval, donor recruitment and sample collection; R.R. performed histopathological analyses; A.S. and T.T. oversaw the design and interpretation of the experimental data and assisted with manuscript editing.

Acknowledgements

The authors thank the SIgN Immunomonitoring platform (Flow and Multiplex Analysis of Proteins), and Biological Resource Centre (BRC) for animal husbandry and veterinary care. We thank Dr. Kim Vanuytsel for helpful discussions regarding CD34⁺ fetal liver cells. Special thanks to Drs. Laurent Renia and Fariyal Ahmed for managing the IAF-PP grant.

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Fetal liver CD34⁺ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Mice

Isolation of Human HSCs

FL-CD34⁺ sorting and cytospin

Human cell transplantation

Cell line-derived xenograft (CDX) tumor model

Humanized mouse tissue isolation and processing

Flow Cytometric analysis of blood and tissue-infiltrating immune cells

Histology

Multiplex Microbead-Based Immunoassay

ELISAs

HLA Typing

Statistical methods

Results

CD34⁺ cells isolated from fetal liver have a distinct phenotype compared to cord blood- CD34⁺

FL-NOG engraft human endothelial and immune cells

Differences in long-term multi-lineage reconstitution of the human immune system in CB- and FL-NOG mice

FL-CD34⁺-transplanted NOG mice develop a distinct GVHD liver pathology

Allogeneic solid tumors are rejected in FL-CD34⁺-reconstituted mice

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Mice

Isolation of Human HSCs

FL-CD34+ sorting and cytospin

Human cell transplantation

Cell line-derived xenograft (CDX) tumor model

Humanized mouse tissue isolation and processing

Flow Cytometric analysis of blood and tissue-infiltrating immune cells

Histology

Multiplex Microbead-Based Immunoassay

ELISAs

HLA Typing

Statistical methods

Results

CD34+ cells isolated from fetal liver have a distinct phenotype compared to cord blood- CD34+

FL-NOG engraft human endothelial and immune cells

Differences in long-term multi-lineage reconstitution of the human immune system in CB- and FL-NOG mice

FL-CD34+-transplanted NOG mice develop a distinct GVHD liver pathology

Allogeneic solid tumors are rejected in FL-CD34+-reconstituted mice

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Fetal liver CD34⁺ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

FL-CD34⁺ sorting and cytospin

CD34⁺ cells isolated from fetal liver have a distinct phenotype compared to cord blood- CD34⁺

FL-CD34⁺-transplanted NOG mice develop a distinct GVHD liver pathology

Allogeneic solid tumors are rejected in FL-CD34⁺-reconstituted mice