PMC

Reprogramming of adult neural stem cells to a monocytic phenotype by ectopic expression of PU.1. Flow cytometry analysis of expression of the hematopoietic marker CD45 (A–F, and J) and the monocyte markers CD63 (G), CD164 (H), and Mac2 (I). The data in J represent mean ± SEM.

Ectopic expression of PU.1 in neural stem cells induces expression of the monocyte marker Iba1. (A–D) Adult neural stem cells were transduced with lentivurs expressing either GFP [lenti-GFP (A and C)] or GFP and HA-tagged PU.1 [lenti-PU.1-GFP (B and D)]. The monocyte marker Iba1 is detected in cells ectopically expressing PU.1 (D), but not in cells transduced with the control vector (C).

Chemotaxis by neural stem cell–derived monocytes. Neural stem cell–derived CD45⁺/GFP⁺ lenti-PU.1-GFP–transduced cells or BV2 monocytes, respectively, placed in the top well migrate toward monocyte chemoattractant protein-1 (MCP-1) in the bottom well in a transwell assay. This is considered a chemotactic response rather than a result of increased motility, because placing MCP-1 in the top well with the CD45⁺/GFP⁺ lenti-PU.1-GFP–transduced cells fails to induce migration to the bottom well. Neural stem cells transduced with control lenti-GFP or cells transduced with lenti-PU.1-GFP that do not express CD45 demonstrate no chemotactic response to MCP-1. The data represent mean ± SEM.

PU.1 reprogrammed neural stem cell–derived cells display monocyte traits. (A and B) Neural stem cell–derived CD45⁺/GFP⁺ lenti-PU.1-GFP–transduced cells were transplanted into the lateral ventricle in E15 mouse embryos. A GFP-labeled cell (shown in the box in A and in higher magnification in B) expresses the monocyte marker Iba1 and displays a morphology indistinguishable from that of a resident microglial cell at 4 d after transplantation. The GFP-labeled cell contains two vacuoles with DAPI⁺-condensed chromatin (asterisks), indicating phagocytosis of cellular debris. The dashed lined delineates the cell's own nucleus. (C) Iba1-expressing PU.1-transduced neurosphere cells in vitro were found to have phagacytotic capacity and to contain condensed DAPI⁺ cellular debris (asterisks). The dashed lined delineates the cell's own nucleus. (Scale bar: 25 μm.)

Transient expression of PU.1 in neural stem cells is sufficient for reprogramming. (A) Over time, an increasing number of neural stem cell–derived cells transduce with lenti-PU.1-GFP lack GFP expression, but are still expressing CD45 (CD45⁺/GFP⁻) at 8 days after transduction (). This population of cells could have arisen in at least two ways; either transduced cells silenced the lentiviral expression of PU.1-HA-GFP but the initial expression of PU.1 was sufficient to activate an endogenous genetic monocyte program, or, alternatively, nontransduced cells were induced to express CD45 by paracrine signals from transduced cells. (B) We analyzed genomic DNA for the presence of integrated viral genetic material. The presence of the viral 5′ LTR sequence was found at similar levels in flow cytometry–isolated CD45⁺/GFP⁻ cells as in CD45⁺/GFP⁺ and CD45⁻/GFP⁺ cells. In contrast, 5′ LTR sequences were detected at levels only marginally above background in CD45⁻/GFP⁻ cells. This indicates that the CD45⁺/GFP⁻ cells were initially transduced and the new monocytic marker profile in these cells was established and stabilized, even though the viral expression of PU.1 was later silenced.