Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.

Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.

Long-term fate of grafted hippocampal neural progenitor cells following ischemic injury.

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long-term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB-Cg-Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP-positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long-term fate of grafted GFP-positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP-positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury.

Age-related ultrastructural changes in spheroids of the adipose-derived multipotent mesenchymal stromal cells from ovariectomized mice.

Introduction: Adipose-derived multipotent mesenchymal stromal cells (ADSCs) are widely used for cell therapy, in particular for the treatment of diseases of the nervous system. An important issue is to predict the effectiveness and safety of such cell transplants, considering disorders of adipose tissue under age-related dysfunction of sex hormones production. The study aimed to investigate the ultrastructural characteristics of 3D spheroids formed by ADSCs of ovariectomized mice of different ages compared to age-matched controls. Methods: ADSCs were obtained from female CBA/Ca mice randomly divided into four groups: CtrlY-control young (2 months) mice, CtrlO-control old (14 months) mice, OVxY-ovariectomized young mice, and OVxO-ovariectomized old mice of the same age. 3D spheroids were formed by micromass technique for 12-14 days and their ultrastructural characteristics were estimated by transmission electron microscopy. Results and Discussion: The electron microscopy analysis of spheroids from CtrlY animals revealed that ADSCs formed a culture of more or less homogeneous in size multicellular structures. The cytoplasm of these ADSCs had a granular appearance due to being rich in free ribosomes and polysomes, indicating active protein synthesis. Extended electron-dense mitochondria with a regular cristae structure and a predominant condensed matrix were observed in ADSCs from CtrlY group, which could indicate high respiratory activity. At the same time, ADSCs from CtrlO group formed a culture of heterogeneous in size spheroids. In ADSCs from CtrlO group, the mitochondrial population was heterogeneous, a significant part was represented by more round structures. This may indicate an increase in mitochondrial fission and/or an impairment of the fusion. Significantly fewer polysomes were observed in the cytoplasm of ADSCs from CtrlO group, indicating low protein synthetic activity. The cytoplasm of ADSCs in spheroids from old mice had significantly increased amounts of lipid droplets compared to cells obtained from young animals. Also, an increase in the number of lipid droplets in the cytoplasm of ADSCs was observed in both the group of young and old ovariectomized mice compared with control animals of the same age. Together, our data indicate the negative impact of aging on the ultrastructural characteristics of 3D spheroids formed by ADSCs. Our findings are particularly promising in the context of potential therapeutic applications of ADSCs for the treatment of diseases of the nervous system.

Relationship of Grafted FGF-2-Overexpressing Neural Stem/Progenitor Cells With the Vasculature in the Cerebral Cortex.

Neural progenitor cells (NPCs) overexpressing fibroblast growth factor 2 (FGF-2) have the distinct tendency to associate with the vasculature and establish multiple proliferative clusters in the perivascular environment after transplantation into the cerebral cortex. Strikingly, the vascular clusters of progenitor cells give rise to immature neurons after ischemic injury, raising prospects for the formation of ectopic neurogenic niches for repair. We investigated the spatial relationship of perivascular clusters with the host vascular structures. FGF-2-GFP-transduced NPCs were transplanted into the intact somatosensory rat cortex. Confocal microscopic analysis revealed that grafted cells preferentially contacted venules at sites with aquaporin-4-positive astrocytic endfeet and avoided contacts with desmin-positive pericytes. Electron microscopic analysis confirmed that grafted cells preferentially made contact with astroglial endfeet, and only a minority of them reached the endothelial basal lamina. These results provide new insights into the fine structural and anatomical relationship between grafted FGF-2-transduced NPCs and the host vasculature.

Ultrastructural study of mouse adipose-derived stromal cells induced towards osteogenic direction.

We investigated the ultrastructural characteristics of mouse adipose-derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB-Cg-Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast-like appearance to having a polygonal osteoblast-like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose-derived stem/stromal cells. Microsc. Res. Tech. 79:557-564, 2016. © 2016 Wiley Periodicals, Inc.