Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking

Abstract

Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseus impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9–GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body following insult or injury. Alternatively, this method might find utility in delivering therapeutic genes for neuroinflammatory conditions.

M2 macrophages, but not M1 macrophages, support megakaryopoiesis by upregulating PI3K-AKT pathway activity

“… 48 , 49 AAV2 viral vectors were produced encoding PI3K shRNA and coexpressing the ZsGreen reporter gene under the control of the MФ-specific AAV2-F4/80-PI3K promoter. 50 , 51 Empty vector under the control of the MФ-specific AAV2-F4/80 promoter was constructed at the same time. AAV2-F4/80-PI3K viral titer of 1.2 × 10 13 vector genomes (vg)/mL, AAV2-F4/80 viral titer of 1.2 × 10 13 vg/mL were used in the study.…”

Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation

“…The 26 G needle was removed and a Hamilton syringe with a 22 S needle gauge (Z15364-8, 100 μL Hamilton, Gastight 1800, needle Gauge 22S) was inserted into the expanded hole created by the 26 G needle. Ten microliters of viral solution (1.8 × 10 13 viral particles/mL AAV2-Cre-DIO- caPpif -eGFP) was slowly injected by free hand into the marrow space of the tibiae (injection technique and viral load adapted from [ Selenica et al, 2016 ]). Contralateral tibiae were injected with 10 μL of sterile PBS solution as an intra-mouse control.…”

CCL2 Overexpression in the Brain Promotes Glial Activation and Accelerates Tau Pathology in a Mouse Model of Tauopathy

“…We have previously reported the impact of CCL2 on microglia polarization in wild-type mice (23,46). Therefore, we aimed to investigate the effect of CCL2 on expression levels of a range of cytokines in a tau animal model.…”