Novel Peripherally Derived Neural-Like Stem Cells as Therapeutic Carriers for Treating Glioblastomas

Abstract

Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary malignant adult brain tumor characterized by extensive invasiveness, heterogeneity, and angiogenesis. Standard treatment options such as radiation and chemotherapy have proven to be only marginally effective in treating GBM because of its invasive nature. Therefore, extensive efforts have been put forth to develop tumor-tropic stem cells as viable therapeutic vehicles with potential to treat even the most invasive tumor cells that are harbored within areas of normal brain. To this end, we discovered a newly described NG2-expressing cell that we isolated from a distinct pericyte subtype found abundantly in cultures derived from peripheral muscle. In this work, we show the translational significance of these peripherally derived neural-like stem cells (NLSC) and their potential to migrate toward tumors and act as therapeutic carriers. We demonstrate that these NLSCs exhibit in vitro and in vivo GBM tropism. Furthermore, NLSCs did not promote angiogenesis or transform into tumor-associated stromal cells, which are concerns raised when using other common stem cells, such as mesenchymal stem cells and induced neural stem cells, as therapeutic carriers. We also demonstrate the potential of NLSCs to express a prototype therapeutic, tumor necrosis factor α-related apoptosis-inducing ligand and kill GBM cells in vitro. These data demonstrate the therapeutic potential of our newly characterized NLSC against GBM. Stem Cells Translational Medicine 2017;6:471-481.

Keywords: Cellular therapy; Glioblastoma; Muscle stem cells; Neural differentiation; Neural-like stem cells.

Figure 1

Isolation of Nestin‐GFP⁺ neural‐like stem cells (NLSCs) from FDB muscle cultures derived from Nestin‐GFP transgenic mice. (A): Unsorted FDB‐derived cell culture is shown in the left panel. NLSCs are green; all cells are counterstained with Hoechst nuclear stain (blue color). The merged picture (left panel) shows the abundance of Nestin‐GFP⁺ cells in the heterogeneous cell mixture derived from FDB muscles. Homogenous population of Nestin‐GFP⁺ NLSCs post FACS is shown in the right panel. NLSCs are green and counterstained with Hoechst nuclear stain (blue color). Scale bars = 100 μm. (B): Representative histograms for FDB culture before and after FACS. Histograms show the purity of the obtained GFP⁺ cell population. Abbreviations: FACS, fluorescence‐activated cell sorting; FDB, flexor digitorum brevis; GFP, green fluorescent protein.

Figure 2

Isolation of GFP⁺ neural‐like stem cells (NLSCs) from Nestin transgenic mice using NGFR (p75) antibody. (A): Labeling of flexor digitorum brevis (FDB) muscle‐derived NLSCs in culture with NGFR (p75) antibody. NLSCs are green owing to expression of GFP; NGFR antibody was detected using Alexa Fluor 647 secondary antibody (red color); all cells were counterstained with Hoechst nuclear stain (blue color). The merged picture shows the expression of NGFR, only on GFP⁺ NLSCs in the heterogeneous cell mixture derived from FDB muscles. This shows that NLSCs from nontransgenic animals can potentially be isolated using nerve growth factor cell surface receptor specific antibody (×20 magnification). (B): Representative histograms for FDB cultures before and after NGFR antibody‐based FACS. Histograms show purity of the obtained NGFR⁺ cell population. (C): Dot plot analysis showed a distinct GFP⁺/NGFR⁺ population. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; FACS, fluorescence‐activated cell sorting; GFP, green fluorescent protein; NGFR, nerve growth factor receptor.

Figure 3

Neural‐like stem cells show tumor‐tropic behavior in vitro and in vivo. (A): Nestin‐GFP⁺ NLSCs in glioblastoma‐conditioned medium and control medium postmigration. NLSCs are green owing to expression of GFP under control of the Nestin promoter. Significant migration of GFP⁺ NLSCs toward G26H2‐conditioned media and U87‐conditioned media was observed when compared with their migration toward control media. Scale bars = 10 μm. (B): Graphical form of migration shown in panel A. Data are expressed as mean ± SEM (n = 3). The results indicated chemotactic behavior of Nestin‐GFP⁺ NLSCs toward cytokines secreted by G26H2 glioblastoma cell line. ∗, p < .05, Student's t test. (C): Fluorescence imaging of brain sections showing that intracranially injected GFP⁺ NLSCs (green) migrate toward orthotropic DsRed⁺ U87 tumors in vivo. Scale bars = 100 μm. (D): Extensive migration of GFP⁺ NLSC to site of DsRed⁺ U87 tumors was observed. Images show presence of migrated NLSCs within the tumor and its infiltrates. Scale bars = 100 μm. Abbreviations: GFP, green fluorescent protein; NLSC, neural‐like stem cells.

Figure 4

NLSCs do not stimulate endothelial tube network formation in vitro or in vivo. (A): Human MSCs (green) and murine MSCs cocultured with HUVECs for 14 days in Matrigel‐coated plates formed endothelial tubular structures. GFP⁺ NLSCs (green) cocultured with HUVECs under the same experimental conditions did not form endothelial tubular structures. The results show that HUVECs were stimulated by MSCs to form vascular structures in vitro, but in contrast, HUVECs were not stimulated to form such structures by NLSC. Scale bars = 100 μm. (B): Matrigel plugs extracted after 2 weeks from nude mice show contrasting vascularization. Matrigel plug containing NLSCs plus HUVECs shows no vascularization, whereas Matrigel plug containing MSCs plus HUVECs shows extensive vascularization. (C): GFP⁺ NLSCs inside the same Matrigel plug are observed under a fluorescence microscope (×4 and ×10 magnifications). Abbreviations: GFP, green fluorescent protein; h, human; HUVEC, human umbilical vein endothelial cell; MSC, mesenchymal stem cells; NLSC, neural‐like stem cells.

Figure 5

NLSCs do not differentiate into tumor‐associated fibroblasts. (A): Expression of αSMA, collagen type 1, and fibroblast‐associated protein cell surface markers by murine MSCs in vitro upon stimulation by TGF‐β‐supplemented media was observed under a fluorescence microscope using immunocytochemistry. Scale bars = 50 μm; ×20 magnification. (B): Absence of αSMA, collagen type 1, and fibroblast‐activated protein‐1 cell surface marker on GFP⁺ NLSCs after stimulation by TGF‐β‐supplemented media. Scale bars = 100 μm; ×10 magnification. Abbreviations: FAP, fibroblast‐associated protein; GFP, green fluorescent protein; m, murine, MSC, mesenchymal stem cells; NLSC, neural‐like stem cells; αSMA, α smooth muscle actin; TGF, transforming growth factor.

Figure 6

NLSCs can be genetically engineered for therapeutic purposes. (A): TRAIL‐mCherry expression by engineered NLSC. NLSCs were transfected with a plasmid encoding TRAIL‐mCherry under the control of the cytomegalovirus promoter. Scale bars = 10 μm. (B): Cytotoxic effect of TRAIL‐expressing NLSCs against U251 cell line measured by the luminescence signal from culture plates. ****, Significant reduction (p < .001) in the luminescence signal from U251 cultures containing engineered NLSCs was observed when compared with control cultures. (C): Cytotoxic effect of TRAIL‐expressing NLSCs against U251 cell line measured by counting the number of viable U251 cells. ***, Significant decrease (p < .001) in the number of viable U251 cells was observed in cultures containing engineered NLSCs when compared with control cultures. (D): Western blotting showed the presence of cleaved PARP, which is an indication of TRAIL‐induced apoptosis in U251 cultures containing engineered NLSCs. Cleaved PARP was not detected in control cultures. Abbreviations: CTRL, control; GBM, glioblastoma; GFP, green fluorescent protein; NLSC, neural‐like stem cells; PARP, poly ADP ribose polymerase; TRAIL, tumor necrosis factor‐(TNF)‐α‐related apoptosis‐inducing ligand.

Figure 7

Pericyte heterogeneity and the origin of NLSCs. We discovered that there are 2 subsets of pericytes that have distinct differentiation potentials. Type 1 pericytes (yellow: Nestin⁻/NG2⁺) appear to differentiate into fat and fibroblasts. In contrast, type 2 pericytes (green: Nestin⁺/NG2⁺) differentiate into muscle fibers, NLSCs, and support angiogenesis. We found that these NLSCs do not differentiate into fibroblasts (Fig. 5). In contrast, heterogeneous populations of MSCs can differentiate into fibroblasts, including tumor‐associated fibroblasts, which can potentially aid tumor cell growth and resistance. Additionally, NLSCs did not contribute to angiogenesis, in contrast to MSCs, as demonstrated in an in vivo plug assay (Fig. 4). Abbreviations: MSCs, mesenchymal stem cells; NLSCs, neural‐like stem cells.