Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

Stem Cell Therapy Enhances Motor Activity of Triceps Surae Muscle in Mice with Hereditary Peripheral Neuropathy.

Impaired motor and sensory functions are the main features of Charcot-Marie-Tooth disease. Mesenchymal stem cell (MSCs) therapy is one of the possible treatments for this disease. It was assumed that MSCs therapy can improve the contractile properties of the triceps surae (TS) muscles in mice with hereditary peripheral neuropathy. Murine adipose-derived mesenchymal stromal cells (AD-MSCs) were obtained for transplantation into TS muscles of FVB-C-Tg(GFPU)5Nagy/J mice. Three months after AD-MSCs transplantation, animals were subjected to electrophysiological investigations. Parameters of TS muscle tension after intermittent high frequency electrical sciatic nerve stimulations were analyzed. It was found that force of TS muscle tension contraction in animals after AD-MSCs treatment was two-time higher than in untreated mice. Normalized values of force muscle contraction in different phases of electrical stimulation were 0.3 ± 0.01 vs. 0.18 ± 0.01 and 0.26 ± 0.03 vs. 0.13 ± 0.03 for treated and untreated animals, respectively. It is assumed that the two-fold increase in TS muscle strength was caused by stem cell therapy. Apparently, AD-MSCs therapy can promote nerve regeneration and partial restoration of muscle function, and thus can be a potential therapeutic agent for the treatment of peripheral neuropathies.

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.

Oligodendrocytes in human induced pluripotent stem cell-derived cortical grafts remyelinate adult rat and human cortical neurons.

Neuronal loss and axonal demyelination underlie long-term functional impairments in patients affected by brain disorders such as ischemic stroke. Stem cell-based approaches reconstructing and remyelinating brain neural circuitry, leading to recovery, are highly warranted. Here, we demonstrate the in vitro and in vivo production of myelinating oligodendrocytes from a human induced pluripotent stem cell (iPSC)-derived long-term neuroepithelial stem (lt-NES) cell line, which also gives rise to neurons with the capacity to integrate into stroke-injured, adult rat cortical networks. Most importantly, the generated oligodendrocytes survive and form myelin-ensheathing human axons in the host tissue after grafting onto adult human cortical organotypic cultures. This lt-NES cell line is the first human stem cell source that, after intracerebral delivery, can repair both injured neural circuitries and demyelinated axons. Our findings provide supportive evidence for the potential future use of human iPSC-derived cell lines to promote effective clinical recovery following brain injuries.

Fibroblast growth factor-2 overexpression in transplanted neural progenitors promotes perivascular cluster formation with a neurogenic potential.

Stem/progenitor cell-based therapies hold promises for repairing the damaged nervous system. However, the efficiency of these approaches for neuronal replacement remains very limited. A major challenge is to develop pretransplant cell manipulations that may promote the survival, engraftment, and differentiation of transplanted cells. Here, we investigated whether overexpression of fibroblast growth factor-2 (FGF-2) in grafted neural progenitors could improve their integration in the host tissue. We show that FGF-2-transduced progenitors grafted in the early postnatal rat cortex have the distinct tendency to associate with the vasculature and establish multiple proliferative clusters in the perivascular environment. The contact with vessels appears to be critical for maintaining progenitor cells in an undifferentiated and proliferative phenotype in the intact cortex. Strikingly, perivascular clusters of FGF-2 expressing cells seem to supply immature neurons in an ischemic environment. Our data provide evidence that engineering neural progenitors to overexpress FGF-2 may be a suitable strategy to improve the integration of grafted neural progenitor cells with the host vasculature thereby generating neurovascular clusters with a neurogenic potential for brain repair.

Adipose-Derived Stem Cells Reduce Lipopolysaccharide-Induced Myelin Degradation and Neuroinflammatory Responses of Glial Cells in Mice.

Brain inflammation is a key event triggering the pathological process associated with many neurodegenerative diseases. Current personalized medicine and translational research in neurodegenerative diseases focus on adipose-derived stem cells (ASCs), because they are patient-specific, thereby reducing the risk of immune rejection. ASCs have been shown to exert a therapeutic effect following transplantation in animal models of neuroinflammation. However, the mechanisms by which transplanted ASCs promote cell survival and/or functional recovery are not fully understood. We investigated the effects of ASCs in in vivo and in vitro lipopolysaccharide (LPS)-induced neuroinflammatory models. Brain damage was evaluated immunohistochemically using specific antibody markers of microglia, astroglia and oligodendrocytes. ASCs were used for intracerebral transplantation, as well as for non-contact co-culture with brain slices. In both in vivo and in vitro models, we found that LPS caused micro- and astroglial activation and oligodendrocyte degradation, whereas the presence of ASCs significantly reduced the damaging effects. It should be noted that the observed ASCs protection in a non-contact co-culture suggested that this effect was due to humoral factors via ASC-released biomodulatory molecules. However, further clinical studies are required to establish the therapeutic mechanisms of ASCs, and optimize their use as a part of a personalized medicine strategy.

Grafted human pluripotent stem cell-derived cortical neurons integrate into adult human cortical neural circuitry.

Several neurodegenerative diseases cause loss of cortical neurons, leading to sensory, motor, and cognitive impairments. Studies in different animal models have raised the possibility that transplantation of human cortical neuronal progenitors, generated from pluripotent stem cells, might be developed into a novel therapeutic strategy for disorders affecting cerebral cortex. For example, we have shown that human long-term neuroepithelial-like stem (lt-NES) cell-derived cortical neurons, produced from induced pluripotent stem cells and transplanted into stroke-injured adult rat cortex, improve neurological deficits and establish both afferent and efferent morphological and functional connections with host cortical neurons. So far, all studies with human pluripotent stem cell-derived neurons have been carried out using xenotransplantation in animal models. Whether these neurons can integrate also into adult human brain circuitry is unknown. Here, we show that cortically fated lt-NES cells, which are able to form functional synaptic networks in cell culture, differentiate to mature, layer-specific cortical neurons when transplanted ex vivo onto organotypic cultures of adult human cortex. The grafted neurons are functional and establish both afferent and efferent synapses with adult human cortical neurons in the slices as evidenced by immuno-electron microscopy, rabies virus retrograde monosynaptic tracing, and whole-cell patch-clamp recordings. Our findings provide the first evidence that pluripotent stem cell-derived neurons can integrate into adult host neural networks also in a human-to-human grafting situation, thereby supporting their potential future clinical use to promote recovery by neuronal replacement in the patient's diseased brain.

EMMPRIN overexpression in SVZ neural progenitor cells increases their migration towards ischemic cortex.

Stimulation of endogenous neurogenesis and recruitment of neural progenitors from the subventricular zone (SVZ) neurogenic site may represent a useful strategy to improve regeneration in the ischemic cortex. Here, we tested whether transgenic overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN), the regulator of matrix metalloproteinases (MMPs) expression, in endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ) could increase migration towards ischemic injury. For this purpose, we applied a lentivector-mediated gene transfer system. We found that EMMPRIN-transduced progenitors exhibited enhanced MMP-2 activity in vitro and showed improved motility in 3D collagen gel as well as in cortical slices. Using a rat model of neonatal ischemia, we showed that EMMPRIN overexpressing SVZ cells invade the injured cortical tissue more efficiently than controls. Our results suggest that EMMPRIN overexpression could be suitable approach to improve capacities of endogenous or transplanted progenitors to invade the injured cortex.

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.

Ultrastructural analysis of murine hippocampal neural progenitor cells in culture.

Despite the great number of studies devoted to neural stem/progenitor cell biology, the ultrastructural characteristics of these cells in vitro have not been fully studied. To determine the fine structure of hippocampal neural progenitor cells (NPCs) in culture, mouse fetal hippocampi (E18) were extracted, dissected, and cells were expanded as adherent monolayer culture. Electron microscopy revealed that NPCs had an immature phenotype, with a high nuclear/cytoplasmic ratio, small and scant organelles, underdeveloped endoplasmic reticulum, and many free ribosomes and polysomes. Our results may contribute to a better understanding of the fine structure and physiology of hippocampal NPCs in vitro.