Bone Marrow-Derived Cell Accumulation in the Spinal Cord Is Independent of Peripheral Mobilization in a Mouse Model of Amyotrophic Lateral Sclerosis

Abstract

Bone marrow-derived cells (BMDCs) are capable of migrating across the blood-brain barrier (BBB) and accumulating in the central nervous system (CNS) when transplanted into recipients conditioned with whole-body irradiation or chemotherapy. We used the chemotherapeutic agents busulfan and treosulfan to condition recipient mice for transplantation with bone marrow (BM) cells isolated from donor mice ubiquitously expressing green fluorescent protein. We attempted to increase the accumulation of BMDCs in the CNS by mobilization of BMDCs using either, or both, granulocyte colony-stimulating factor (GCSF) or plerixafor (AMD3100). We also used several concentrations of busulfan. We hypothesized that higher concentrations of busulfan and BMDC mobilization would increase numbers of GFP⁺ cells in the CNS. The doses of busulfan employed (60-125 mg/kg) all resulted in high levels of sustained chimerism (>85% 1 year post-transplant) in both the blood and BM of wild-type (WT) mice and an amyotrophic lateral sclerosis (ALS) mouse model. Moreover, cells accumulated within the CNS in a dose-, time-, and disease-dependent manner. Conditioning with the hydrophilic busulfan analog treosulfan, which is unable to cross the BBB efficiently, also resulted in a high degree of BM chimerism. However, few GFP⁺ BMDCs were found within the CNS of WT or ALS mice of treosulfan-conditioned mice. Mobilization of BMDCs into the circulation using GCSF and/or AMD3100 did not lead to increased accumulation of GFP⁺ BMDCs within the CNS of WT or ALS mice. Weekly analysis of BMDC accumulation revealed that BMDCs accumulated more rapidly and to a greater extent in the CNS of ALS mice conditioned with a high dose (125 mg/kg) of busulfan compared to a lower dose (80 mg/kg). The number of GFP⁺ BMDCs in the CNS labeling with the proliferation marker Ki67 increased in parallel with BMDC accumulation within the CNS. Our results indicate that establishment of high levels of blood and BM chimerism alone is not sufficient to induce BMDC accumulation within the CNS and that CNS conditioning is a crucial requirement for BMDC accumulation to occur. Moreover, it appears that proliferation of BMDCs that infiltrate the CNS is partly responsible for cell accumulation in busulfan-conditioned ALS mice.

Keywords: AMD3100; amyotrophic lateral sclerosis; bone marrow-derived cells; busulfan; central nervous system; granulocyte colony-stimulating factor; monocyte; treosulfan.

Figure 1

High levels of chimerism are rapidly established and sustained in BU-conditioned mice. (A) Weekly flow cytometric quantification of GFP⁺ cells within the blood in mutant SOD1 mice conditioned with 125 mg/kg of BU and transplanted with GFP⁺ donor bone marrow (BM) cells. (B) Flow cytometric quantification of GFP⁺ cells within the blood and BM 1-year post-bone marrow transplantation in wild-type mice conditioned with the indicated dose of BU. Data = mean ± SEM for n = 3 mice.

Figure 2

Bone marrow-derived cells accumulate in the lumbar spinal cord of BU-conditioned mice in a time- and dose-dependent manner. (A) Quantification of GFP⁺ donor cells within the lumbar spinal cord 1 year post-bone marrow transplantation (BMT) in wild-type (WT) mice conditioned with the indicated dose of BU (milligrams per kilogram). (B) Quantification of GFP⁺ donor cells within the lumbar spinal cord in WT mice conditioned with 80 mg/kg of BU and collected at the indicated times post-BMT. Data = mean ± SEM for n = 3 mice/group; data were analyzed by ANOVA with p < 0.05.

Figure 3

Bone marrow-derived cells accumulate to a much greater extent in the lumbar spinal cord of BU-conditioned mutant SOD1 (mSOD) mice compared to wild-type (WT) controls. Immunohistochemical analysis of lumbar spinal cord sections from late stage mSOD mice and age-matched WT controls that were conditioned with 125 mg/kg BU and transplanted with GFP⁺ donor bone marrow cells at 6–8 weeks of age. GFP⁺ BMDCs are shown in green. Results are representative images from n ≥ 3 mice.

Figure 4

High levels of blood and bone marrow (BM) chimerism are achieved in TREO-conditioned mice. Flow cytometric quantification of GFP⁺ cells within the blood and BM in late stage mutant SOD1 mice that were conditioned with 4,500 or 6,000 mg/kg TREO and transplanted with GFP⁺ donor BM cells at 6–8 weeks of age. Data = mean ± SEM for n ≥ 3 mice/group; data were analyzed by a Student’s t-test with p < 0.05.

Figure 5

Mobilization of bone marrow-derived cells (BMDCs) into the circulation does not increase BMDC accumulation within the lumbar spinal cord of mutant SOD1 (mSOD) mice and wild-type (WT) controls. (A) Flow cytometric analysis of peripheral blood following mobilization treatments in WT mice. Results are representative images from n = 3 mice. (B) The 6- to 8-week-old WT mice were conditioned with 80 mg/kg of BU and transplanted with GFP⁺ donor bone marrow (BM) cells. Chimerism was allowed to establish for 5 weeks prior to mobilization treatment. GFP⁺ cells were quantified in lumbar spinal cord sections at the indicated timepoints. Data = mean ± SEM for n ≥ 3 mice/treatment. (C,D) The 6- to 8-week-old mSOD mice were conditioned with 80 mg/kg of BU and transplanted with GFP⁺ donor BM cells. Chimerism was allowed to establish for 5 weeks (C) or 3 days (D) prior to mobilization treatment. GFP⁺ cells were quantified in lumbar spinal cord sections at disease end stage. Data = mean ± SEM for n ≥ 4 mice/treatment.

Figure 6

Bone marrow-derived cells accumulate more rapidly and to a greater extent in mutant SOD1 (mSOD) mice conditioned with a high dose of BU compared to a lower dose. (A) Weekly quantification of GFP⁺ cells within the lumbar spinal cord in mSOD mice conditioned with either 80 or 125 mg/kg BU and transplanted at 15 weeks of age. Data = mean ± SEM for n = 3 mice/timepoint. (B) Weekly quantification of GFP⁺ cell morphology within the lumbar spinal cord in mSOD mice conditioned with 80 mg/kg BU and transplanted at 15 weeks of age. Data = mean ± SEM for n = 3 mice/timepoint. (C) Weekly quantification of GFP⁺ cell morphology within the lumbar spinal cord in mSOD mice conditioned with 125 mg/kg BU and transplanted at 15 weeks of age. Data = mean ± SEM for n = 3 mice/timepoint. (D) Weekly quantification of Ki67⁺ cells and GFP⁺Ki67⁺ cells within the lumbar spinal cord in mSOD mice conditioned with either 80 or 125 mg/kg BU and transplanted at 15 weeks of age. Data = mean ± SEM for n = 3 mice/timepoint. (E) Weekly quantification of Iba1⁺ cells within the lumbar spinal cord in mSOD mice conditioned with either 80 or 125 mg/kg BU and transplanted at 15 weeks of age. Data = mean ± SEM for n = 3 mice/timepoint. (F) Immunohistochemical analysis of lumbar spinal cord sections from late stage mSOD mice that were conditioned with 125 mg/kg BU and transplanted with GFP⁺ donor BM cells at 15 weeks of age. Mice were given IP injections of 50 mg/kg 5-ethynyl-2′-deoxyuridine (EdU) 48 and 24 h prior to collection to label proliferating cells. Nuclei are shown in blue, EdU in red, and GFP⁺ cells in green. Results are representative images from n = 3 mice. Data were analyzed by ANOVA, and post hoc tests using the Tukey–Kramer method were used. * represents a significant difference between 80 and 125 mg/kg treatment groups with p < 0.01; ŧ represents a significant difference from the preceding timepoint with p < 0.01; Ŧ represents a significant difference from the preceding timepoint with p = 0.02.