Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses

Hirokazu Ohtaki, Joni H Ylostalo, Jessica E Foraker, Andrew P Robinson, Roxanne L Reger, Seiji Shioda, Darwin J Prockop, Hirokazu Ohtaki, Joni H Ylostalo, Jessica E Foraker, Andrew P Robinson, Roxanne L Reger, Seiji Shioda, Darwin J Prockop

Abstract

Human mesenchymal stromal cells (hMSCs) were injected into the hippocampus of adult mice 1 day after transient global ischemia. The hMSCs both improved neurologic function and markedly decreased neuronal cell death of the hippocampus. Microarray assays indicated that ischemia up-regulated 586 mouse genes. The hMSCs persisted for <7 days, but they down-regulated >10% of the ischemia-induced genes, most of which were involved in inflammatory and immune responses. The hMSCs also up-regulated three mouse genes, including the neuroprotective gene Ym1 that is expressed by activated microglia/macrophages. In addition, the transcriptomes of the hMSC changed with up-regulation of 170 human genes and down-regulation of 54 human genes. Protein assays of the hippocampus demonstrated increased expression in microglia/macrophages of Ym1, the cell survival factor insulin-like growth factor 1, galectin-3, cytokines reflective of a type 2 T cell immune bias, and the major histocompatibility complex II. The observed beneficial effects of hMSCs were largely explained by their modulation of inflammatory and immune responses, apparently by alternative activation of microglia and/or macrophages.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effects of tCCAO with and without injection of hMSCs into DG of hippocampus of mice. (A) Within 5 min after cell injection [hMSCs (+)] into the DG of IC mice without tCCAO the cells were found in the hippocampus (Hip), neocortex (Ctx), striatum (Str), and cerebellum (Ce) (n = 6 hippocampi from three mice). (B) Assays by real-time PCR for hAlu sequences after injection of hMSCs into ID or IC mice with or without previous tCCAO (n = 6 hippocampi). The survival of hMSCs in ID mice was not statistically different from that in IC mice. (C) Typical labeling for human cells (anti-HuNu) in the hippocampus 4 days after tCCAO (arrows). (D) Higher-magnification images of C indicate the HuNu label in nuclei (DAPI; blue). (E) Neurologic deficit (ND) scores were evaluated by open-field behavior test on day 1 (pretreatment) and on days 2, 3, and 4 after tCCAO. (F) Typical images of FJB staining for degenerating neurons in the hippocampus CA1 region (arrows). (G) Quantitation of FJB-stained cells (n = 6 mice; P < 0.05) indicated decreased death of neurons in mice treated with hMSCs 4 days after tCCAO.
Fig. 2.
Fig. 2.
Microarray assays of hippocampi 2 days after tCCAO and 1 day after injection of HBSS or hMSCs. (A) Effects of ischemia on the mouse transcriptome. Signal intensities (SI) were compared between samples from uninjured/HBSS and ischemia/HBSS mice. (B) Venn diagram of 586 mouse genes up-regulated by ischemia, eight genes further up-regulated by injection of hMSCs, and 80 genes down-regulated by the hMSCs. (C) Venn diagram of 41 mouse genes down-regulated by ischemia and one of the down-regulated genes up-regulated by injection of hMSCs. (D) Effects of hMSCs on the transcriptome of mice after tCCAO. The hMSCs produced up-regulation of only three mouse genes (Lyz, lysozyme; Ym1, also known as Chi313; and Tgbi, TGFβ induced protein). The hMSCs produced down-regulation of 80 mouse genes. The most common GO terms are indicated. (E) Venn diagram of 189 human genes (170 nonredundant) up-regulated in hMSCs by the ischemic environment. (F) Venn diagram of 57 human genes (54 non-cross-hybridizing) down-regulated by the ischemic environment. The genes were filtered for cross-hybridization as indicated in SI Methods. (G) Effects of the ischemic microenvironment on the hMSC transcriptome.
Fig. 3.
Fig. 3.
Injection of hMSCs into hippocampus after tCCAO modified the immune response to a Th2 immune bias. Mouse-specific ELISAs were performed (n = 4–6 mice from each group). (A) Increase in the level of IGF-1 in the hippocampus (n = 5–6; P < 0.05). (B and C) Changes in the IL-4/IFNγ and IL-4/TNFα ratios in the hippocampus indicated a Th2/Th1 response (P < 0.05). (D) IGF-1 (red) coexpressed (arrows) with the microglial and macrophages marker (CD11b, green) in the hippocampus 4 days after tCCAO and 3 days after the injection of hMSCs.
Fig. 4.
Fig. 4.
Injection of hMSCs increased the levels of Gal-3 and numbers of Gal-3 (+) microglia/macrophages 4 days after tCCAO. (A) Mouse-specific Gal-3 ELISA indicated increased levels in hippocampus of the ischemia/hMSCs mice (n = 5 to 6; **, P < 0.01). (B) Increased levels of Gal-3 in the CSF (n = 8–10; *, P < 0.05). (C) Colabeling (arrows) in the hippocampus of Gal-3 (+) cells (green) with markers for microglia/macrophages (F4/80 and CD11b, red). (D) Gal-3 (+) cells (green) in close association with hMSCs (HuNu, red). (E) Higher magnification to demonstrate that Gal-3 (+) cells (arrowheads) were not colabeled with HuNu (arrows).
Fig. 5.
Fig. 5.
Injection of hMSCs after tCCAO increased APC-like features of Gal-3 (+) cells and caused alternative activation of the microglia and/or macrophages in hippocampus. (A) Cells labeled with the APC marker MHC II (green) and with hMSCs (HuNu; red) were seen near the site of injection (Inj.), but the cells were not colabeled. The images were from immediately beneath the DG (above the double arrows). (B) Injection of tCCAO mice with hMSCs increased cells colabeled with MHC II (green) and Gal-3 (red). (C) In tCCAO mice injected with hMSCs, some Gal-3 (+) cells (purple) were colabeled (arrows) with both IGF-1 (green) and MHC II (red). (D) Some MHC II (+) cells (red) near the injection site (Inj.) colabeled (arrows) with Ym1 (green). (E) Injection of hMSCs into tCCAO mice increased number of cells labeled with Ym1 (green) (arrows). (F) Western blots confirmed that injection of hMSCs increased levels of Ym1 in the hippocampi 4 days after tCCAO. The pretreatment control was taken 1 day after tCCAO but before injection of hMSCs. (G) Densitometry of the Western blots in F.
Fig. 6.
Fig. 6.
Schematic illustration of the effects of hMSCs on ischemia produced by tCCAO (see Discussion).

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