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Synergy of Endothelial and Neural Progenitor Cells From Adipose-Derived Stem Cells to Preserve Neurovascular Structures in Rat Hypoxic-Ischemic Brain Injury


Synergy of Endothelial and Neural Progenitor Cells From Adipose-Derived Stem Cells to Preserve Neurovascular Structures in Rat Hypoxic-Ischemic Brain Injury

Yuan-Yu Hsueh et al. Sci Rep.

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Perinatal cerebral hypoxic-ischemic (HI) injury damages the architecture of neurovascular units (NVUs) and results in neurological disorders. Here, we differentiated adipose-derived stem cells (ASCs) toward the progenitor of endothelial progenitor cells (EPCs) and neural precursor cells (NPCs) via microenvironmental induction and investigated the protective effect by transplanting ASCs, EPCs, NPCs, or a combination of EPCs and NPCs (E+N) into neonatal HI injured rat pups. The E+N combination produced significant reduction in brain damage and cell apoptosis and the most comprehensive restoration in NVUs regarding neuron number, normal astrocytes, and vessel density. Improvements in cognitive and motor functions were also achieved in injured rats with E+N therapy. Synergistic interactions to facilitate transmigration under in vitro hypoxic microenvironment were discovered with involvement of the neuropilin-1 (NRP1) signal in EPCs and the C-X-C chemokine receptor 4 (CXCR4) and fibroblast growth factor receptor 1 (FGFR1) signals in NPCs. Therefore, ASCs exhibit great potential for cell sources in endothelial and neural lineages to prevent brain from HI damage.


Figure 1
Figure 1. After endothelial growth medium treatment and laminar shear stress stimulation, the EPCs differentiated from human ASCs were aligned in parallel to the direction of fluid flow and showed an increase in the endothelial markers Flk-1 and vWF (n = 15).
(a) Endothelial functions exhibited increased tube-like structures on Matrigel (n = 10) and uptake of DiI-labeled acLDL (n = 10). (b) Neural differentiation was induced by forming free-floating spheres on the chitosan-coated surface. Spheres expressed the specific differentiation markers nestin and GFAP for neural lineages (n = 13). (c) Immunofluorescent staining showed an increase in nestin, NF-H, and GFAP in the spheres (n = 10). (d) Scale bar in phase image: 200 μm. Scale bar in fluorescent image: 50 μm. Data are represented as mean ± SD. *p < 0.05 compared to undifferentiated ASCs.
Figure 2
Figure 2. Hypoxic-ischemic (HI) injury was created in neonatal rats on postnatal day 7, and the brain was harvested 7 days after injury (n = 4).
(a) Prevention of brain tissue loss was measured by TTC staining after different specified cell therapies (n = 5). (b) Nissl staining revealed that the application of EPCs or NPCs alone minimally prevented brain loss compared to injection of the original ASCs (n = 5). (c) The combination treatment of EPCs and NPCs (E+N) further improved the therapeutic effect in live neurons. Cell apoptosis (brown color) in HI-injured brains was observed with TUNEL staining (n = 5). (d) The E+N combined treatment showed the best outcome in protecting the brain cells from death after HI injury. *p < 0.05 compared to PBS-injected rats.
Figure 3
Figure 3. Improvements in neurovascular structures in HI-injured brain were evaluated with specific staining for vessels (RECA, n = 8), neurons (NeuN, n = 7), and astrocytes (GFAP, n = 7) in the cortex and hippocampus.
(a) The microvascular structure of the injured hemisphere of HI brains was measured by vessel density in the RECA-stained images after different specified cell therapies. (b) Cortical vessel density was increased after the transplantation of ASCs, EPCs, NPCs, or E+N combined therapy. However, the vessel density in the hippocampus was increased when treated with EPCs or the E+N combination. Increases in NeuN (+) cells were observed in the cortex and hippocampus of rats treated with ASCs, EPCs, NPCs, or the E+N combination. (c) After HI injury, staining with GFAP showed reactive astrocyte morphology without cell therapy. (d) Injection of ASCs, NPCs, or the E+N combination inhibited astrocyte activation, but this was not observed with EPC treatment. Scale bar: 100 μm. *p < 0.05 compared to PBS-injected rats.
Figure 4
Figure 4. After transplantation of EPCs and NPCs derived from human ASCs, the engraftment of differentiated cells was identified with double staining (arrowheads) of human chromatin (hChromatin, green) and by specific antibodies against RECA (n = 5), NeuN (n = 5), and GFAP (n = 5) (red).
(a) The E+N combined therapy promoted endogenous neurogenesis as indicated by increased nestin staining specific for rat neural stem cells, but not human nestin. (b) The combination of E+N also increased angiogenesis as demonstrated by increased isolectin IB4 staining (n = 5). (c) Scale bar: 50 μm.
Figure 5
Figure 5. Cognitive function was evaluated using the Morris water maze after animals received different cell transplantations.
The HI-injured rats showed impairment in learning ability, which was improved by all cell therapies (n = 9). (a) No significant differences in normalized time were found among the various treatments in learning ability. In memory function, the numbers of platform crosses were significantly increased in rats that received the E+N combined treatment (n = 9). (b) Motor function was measured by grip strength and showed improvements in rats with ASCs, EPCs, or the E+N combined treatment (n = 9). (c) *p < 0.05 compared to PBS-injected rats.
Figure 6
Figure 6. The combination of EPCs and NPCs increased transmigration in a Boyden chamber under normoxic conditions, and the application of the hypoxia mimetic reagent DFO further promoted the mobility in the E+N group (n = 5).
(a) Different types of cells with or without DFO treatment were harvested to assess the potential gene expression in angiogenesis and neurogenesis signaling pathways for ANG1, ANG2, NRP1, CXCR4, and FGFR1 (n = 3). (b) Specific inhibitions of angiogenic (NRP1 inhibiting peptide, DG2) and neurogenic (AMD3100, a CXCR4 inhibitor and SU5402, an FGFR1 inhibitor) signaling were applied to EPCs or NPCs (n = 5). (c) Synergistic interactions were originated from EPCs via NRP1 signaling and from NPCs by CXCR4 and FGFR1 signaling pathways. Scale bar: 200 μm. *p < 0.05 compared to undifferentiated ASCs under normoxia. #p < 0.05 compared to undifferentiated ASCs under hypoxia.

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