Stem cell transplantation therapy has emerged as a potential treatment for ischemic stroke and other neurodegenerative diseases. Effective delivery of exogenous cells and homing of these cells to the lesion region, however, have been challenging issues that hinder the efficacy and efficiency of cell-based therapy. In the present investigation, we tested a delayed treatment of noninvasive and brain-targeted intranasal delivery of bone marrow mesenchymal stem cells (BMSCs) in a mouse focal cerebral ischemia model. The investigation tested the feasibility and effectiveness of intranasal delivery of BMSCs to the ischemic cortex. Hypoxia preconditioning (HP) of BMSCs was performed before transplantation in order to promote their survival, migration, and homing to the ischemic brain region after intranasal transplantation. Hoechst dye-labeled normoxic- or hypoxic-pretreated BMSCs (1 × 10(6) cells/animal) were delivered intranasally 24 h after stroke. Cells reached the ischemic cortex and deposited outside of vasculatures as early as 1.5 h after administration. HP-treated BMSCs (HP-BMSCs) showed a higher level of expression of proteins associated with migration, including CXC chemokine receptor type 4 (CXCR4), matrix metalloproteinase 2 (MMP-2), and MMP-9. HP-BMSCs exhibited enhanced migratory capacities in vitro and dramatically enhanced homing efficiency to the infarct cortex when compared with normoxic cultured BMSCs (N-BMSCs). Three days after transplantation and 4 days after stroke, both N-BMSCs and HP-BMSCs decreased cell death in the peri-infarct region; significant neuroprotection of reduced infarct volume was seen in mice that received HP-BMSCs. In adhesive removal test of sensorimotor functional assay performed 3 days after transplantation, HP-BMSC-treated mice performed significantly better than N-BMSC- and vehicle-treated animals. These data suggest that delayed intranasal administration of stem cells is feasible in the treatment of stroke and hypoxic preconditioning of transplanted cells, significantly enhances cell's homing to the ischemic region, and optimizes the therapeutic efficacy.

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O2) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K+ channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K+ currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K+ channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK397 and FAK576/577, and this effect was antagonized by blocking K+ channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.

SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1 ؊/؊ mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1 ؊/؊ mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1 ؉/؉ , ؉/؊ , and ؊/؊ mice. SIRT1 ؊/؊ ESCs formed fewer mature blast cell colonies. Replated SIRT1 ؊/؊ blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1 ؊/؊ -derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1 ؊/؊ ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased ␤-H1 globin, ␤-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1 ؊/؊ ESC differentiation deficiencies. SIRT1 ؊/؊ yolk sacs manifested fewer primitive erythroid precursors. SIRT1 ؊/؊ and SIRT1 ؉/؊ adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. (Blood. 2011;117(2):440-450) IntroductionMouse embryonic stem cells (ESCs) are pluripotent with capacity for unlimited self-renewal or differentiation into endoderm, ectoderm, and mesoderm. Self-renewal behavior in vitro is sustained with leukemia inhibitory factor (LIF). 1 With removal of LIF and in the absence of feeder layer cells, ESCs grow into spheres termed embryoid bodies (EBs), which generate hematopoietic and endothelial progeny recapitulating development of those populations in the yolk sac. 2 Hemangioblasts generate blast colonies in vitro displaying hematopoietic and endothelial potential. 3 The ESC/EB system provides a powerful in vitro model to explore cellular and molecular events that specify lineage choice and hematopoietic commitment.Sirtuins, or Sir2 family proteins, are conserved from bacteria to humans. 4 Sir2 modulates longevity and aging in yeast, Caenorhabditis elegans, and Drosophila. 5 Mammalian homologs of Sir2 encompass a family of 7 proteins (SIRT1-SIRT7), among which SIRT1 is the closest human homolog of the yeast Sir2 protein. 4 SIRT1 deacetylates proteins, including p53 and FOXO transcription factors, and plays many key functions including energy metabolism, differentiation, aging, and tumor suppression. [6][7][8][9][10] SIRT1 is expressed at high levels in mouse embryos with the highest SIR2␣ mRNA expression is embryonic day (E) 4.5 embryos. Although expression is down-regulated during subsequent embryogenesis, high level expression rema...

Bone marrow-derived mesenchymal stem cells (BMSCs) have shown great promise for ischemic tissue repair. However, poor viability of transplanted BMSCs within ischemic tissues has limited their therapeutic potential. Apelin, an endogenous peptide, whose level is elevated following ischemia, has been shown to enhance survival of cardiomyocytes and neuronal cells during ischemia. We hypothesized that apelin-13 protects BMSCs from apoptotic death. In this paper we determined the potential mechanism of apelin-13 effects using cultured BMSCs from adult rats. Apoptosis was induced by the specific apoptotic insult serum deprivation (SD) for up to 36 hrs. Apoptotic cell death was measured using immunostaining and Western blotting in the presence and absence of apelin-13 (0.1 to 5.0 nM) co-applied during SD exposure. SD-induced apoptosis was significantly reduced by apelin-13 in a concentration-dependent manner. SD-induced mitochondrial depolarization, cytochrome c release, and caspase-3 activation were largely prevented by apelin-13. The apelin-13 anti-apoptotic effects were blocked by inhibiting the MAPK/ERK1/2 and PI3K/Akt signaling pathways. Taken together, our findings indicate that apelin-13 is a survival factor for BMSCs and its anti-apoptotic property may prove to be of therapeutic significance in terms of exploiting BMSC-based transplantation therapy.

Wnt signaling is a conserved pathway involved in expansion of neural progenitors and lineage specification during development. However, the role of Wnt signaling in the post-stroke brain has not been well-elucidated. We hypothesized that Wnt-3a would play an important role for neurogenesis and brain repair. Adult male mice were subjected to a focal ischemic stroke targeting the sensorimotor cortex. Mice that received Wnt-3a (2 µg/kg/day, 1 h after stroke and once a day for the next 2 days, intranasal delivery) had reduced infarct volume compared to stroke controls. Wnt-3a intranasal treatment of seven days upregulated the expression of brain-derived growth factor (BDNF), increased the proliferation and migration of neuroblasts from the subventricular zone (SVZ), resulting in increased numbers of newly formed neurons and endothelial cells in the peri-infarct zone. Both the molecular and cellular effects of Wnt-3a were blocked by the Wnt specific inhibitors XAV-939 or Dkk-1. In functional assays, Wnt-3a treatment enhanced the local cerebral blood flow (LCBF) in the peri-infarct, as well as improved sensorimotor functions in a battery of behavioral tests. Together, our data demonstrates that the Wnt-3a signaling can act as a dual neuroprotective and regenerative factor for the treatment of ischemic stroke.