Endogenous Neural Stem Cell-induced Neurogenesis after Ischemic Stroke: Processes for Brain Repair and Perspectives.

Ischemic stroke is a very common cerebrovascular accident that occurred in adults and causes higher risk of neural deficits. After ischemic stroke, patients are often left with severe neurological deficits. Therapeutic strategies for ischemic stroke might mitigate neuronal loss due to delayed neural cell death in the penumbra or seek to replace dead neural cells in the ischemic core. Currently, stem cell therapy is the most promising approach for inducing neurogenesis for neural repair after ischemic stroke. Stem cell treatments include transplantation of exogenous stem cells but also stimulating endogenous neural stem cells (NSCs) proliferation and differentiation into neural cells. In this review, we will discuss endogenous NSCs-induced neurogenesis after ischemic stroke and provide perspectives for the therapeutic effects of endogenous NSCs in ischemic stroke. Our review would inform future therapeutic development not only for patients with ischemic stroke but also with other neurological deficits.

Effects of environmental stressors on stem cells.

Environmental toxicants are ubiquitous, and many are known to cause harmful health effects. However, much of what we know or think we know concerning the targets and long-term effects of exposure to environmental stressors is sadly lacking. Toxicant exposure may have health effects that are currently mischaracterized or at least mechanistically incompletely understood. While much of the recent excitement about stem cells (SCs) focuses on their potential as therapeutic agents, they also offer a valuable resource to give us insight into the mechanisms and risks of toxicant effects. Not only as a response to the increasing ethical pressure to reduce animal testing, SC studies allow us valuable insight into the true effects of human exposure to environmental stressors under controlled conditions. We present a review of the history of publications on the effects of environmental stressors on SCs, followed by a consolidation of the literature over the past five years on a subset of key environmental stressors of importance to human health and their effects on both embryonic and tissue SCs. The review will make constructive suggestions as to areas of toxicant research where further studies are needed, as well as making indications of the potential utility for advancing knowledge and directing research on environmental toxicology.

Hypoxic Stress Forces Irreversible Differentiation of a Majority of Mouse Trophoblast Stem Cells Despite FGF4.

Hypoxic, hyperosmotic, and genotoxic stress slow mouse trophoblast stem cell (mTSC) proliferation, decrease potency/stemness, and increase differentiation. Previous reports suggest a period of reversibility in stress-induced mTSC differentiation. Here we show that hypoxic stress at 0.5% O2 decreased potency factor protein by ∼60%-90% and reduced growth to nil. Hypoxia caused a 35-fold increase in apoptosis at Day 3 and a 2-fold increase at Day 6 above baseline. The baseline apoptosis rate was only 0.3%. Total protein was never less than baseline during hypoxic treatment, suggesting 0.5% O2 is a robust, nonmorbid stressor. Hypoxic stress induced ∼50% of trophoblast giant cell (TGC) differentiation with a simultaneous 5- to 6-fold increase in the TGC product antiluteolytic prolactin family 3, subfamily d, member 1 (PRL3D1), despite the presence of fibroblast growth factor 4 (FGF4). Hypoxia-induced TGC differentiation was also supported by potency and differentiation mRNA marker analysis. FGF4 removal at 20% O2 committed cell fate towards irreversible differentiation at 2 days, with similar TGC percentages after an additional 3 days of culture under potency conditions when FGF4 was readded or under differentiation conditions without FGF4. However, hypoxic stress required 4 days to irreversibly differentiate cells. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation limit total available stem cell population. Were mTSCs to respond to stress in a similar mode in vivo, miscarriage might occur as a result, which should be tested in the future.

Translating stem cell research from the bench to the clinic: a need for better quality data.

Stem cell therapies show great medical promise, but few new products have made it into the marketplace. The translation of stem and other cell therapies faces not only challenges associated with research and development, but also the challenges of investment funding and regulatory approval. Regulators and investors alike appear to be voicing the same concerns: they see (1) insufficient high-quality data to provide confidence regarding the claims of medical benefit, (2) an insufficient understanding of the mechanism of action, and (3) a lack of identification of essential characteristics for product release criteria and for assuring reproducibility in manufacturing. The ensuing frustration on the part of researchers and developers may be the result of failure to fully comprehend what is required to assure that confidence.

Stem cell standardization.

Stem cell research promises, despite the uncertainty that still affects many of its technological aspects, to significantly impact a broad range of life science domains (from drug discovery to innovative cell treatments). A decade of intensive research has produced only fragmented knowledge and unsubstantiated options for innovative treatments to be delivered in the clinical setting. Therapeutic applicability can only be realized or, indeed discarded, based on investigations that adopt more rigorous procedures and strategies.

Intestinal epithelial cells in vitro.

Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the difficulty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the current status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profiles of the intestinal cellular differentiation.

Mitochondria: determinants of stem cell fate?

Stem cells are traditionally classified as being either embryonic stem cells (ESCs) or somatic stem cells. Such a designation has now become blurred by the advent of ostensibly pluripotent cells derived from somatic cells, referred to as induced pluripotent stem cells. Mitochondria are the membrane bound organelles that provide the majority of a cell's chemical energy via their production of adenosine triphosphate. Mitochondria are also known to be vital components in many cell processes including differentiation and apoptosis. We are still remarkably uninformed of how mitochondrial function affects stem cell behavior. Reviewed evidence suggests that mitochondrial function and integrity affect stem cell viability, proliferative and differential potential, and lifespan. Mitochondrial status therefore has profound and as yet unexamined implications for the current drive to develop induced pluripotent stem cells as a therapeutic resource.

Mitochondrial dysfunction in a neural cell model of spinal muscular atrophy.

Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC-34 cells to disease levels. Smn siRNA knock-down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock-down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn-depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target.

Survival motor neuron protein regulates apoptosis in an in vitro model of spinal muscular atrophy.

Progressive spinal muscular atrophy (SMA), the most prevalent hereditary lower motor neuron disease, is caused by mutations in the telomeric copy of the survival of motor neuron (SMN1) gene. Unlike other cells, lower motor neurons cannot tolerate low levels of smn protein. However, it is unclear as to the nature of the cell death involved. There is evidence that lower motor neurons undergo apoptosis in SMA, leading to muscle weakness and wasting. This study investigated whether SMN1 regulation in a motor neuron model affected indices of apoptotic cell death. Decreased smn expression in neuroblastoma hybrid (NSC-34) cell lines by small interfering RNA (siRNA) was demonstrated at the mRNA and protein level. Smn-depleted cells showed elevated caspase-3 activity, decreased cell viability and increased percentage of TUNEL positive cells. Conversely, NSC-34 cell smn overexpression by adenoviral gene transfer decreased staurosporine-induced caspase-3 elevation and mitigated induced cell toxicity as assessed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. However, increased smn expression by itself did not increase cell viability. These data suggest not only that decreased smn levels increase apoptosis in an in vitro model of SMA, but also that increased smn can protect against neural injury.

Blood stem cells and non-hematological clinical practice: pragmatics before therapeutics.

There is considerable interest in biological sources for replacement, repair, as well as vascularization of tissue. The remarkable properties of blood stem cells encourage interest in their therapeutic potential. But what are these properties, and how do they influence their clinical potential and the advisability of stem cell use as a therapeutic resource? Rational assessment of the significance of in vitro and animal in vivo data should precede the rush from the bench to the bedside. Basic stem cell research is rife with examples where the truth of the subsequently demonstrated mechanism is stranger than the initial interpretation proved fiction. This review will assess tissue contribution by different blood related stem cells, differing possible mechanisms underlying observed repair phenomena, and consider the potency and pitfalls of stem cell therapeutics.

Systemic nicotine alters whole-body fat utilization in female rats.

Cigarette smoking produces weight suppression in humans that often rebounds following smoking cessation. Nicotine (NIC) administration produces similar effects in rats. While changes in food intake are thought to account for some of the body weight changes, few reports have investigated how NIC affects whole-body metabolism. In the present study, measures of respiratory quotient (RQ) and energy expenditure (EE) were used to investigate metabolic changes that may contribute to NIC's effects on body weight. Female rats (n=46) were implanted for 14 days with subcutaneous Alzet minipumps containing NIC (6 mg/kg/day) or its vehicle. One-hour metabolic test sessions occurred four times: 2 and 12 days after pump implant and 2 and 8 days after pump removal. NIC initially suppressed body weight gain, followed by steady recovery that was briefly exaggerated after withdrawing NIC. Daily food intake was acutely suppressed by NIC and acutely potentiated after NIC cessation. RQ, but not EE, was suppressed by NIC 2 days after pump implant indicating increased fat utilization. Conversely, RQ was increased 2 days after pump removal signaling increased fat storage. These findings indicate that acute changes in whole-body metabolism may contribute to the weight modulating effects of NIC.

Differential responses of human neural and hematopoietic stem cells to ethanol exposure.

The mechanisms underlying fetal developmental defects caused by maternal ethanol (EtOH) consumption remain unclear. The symptoms of fetal alcohol syndrome (FAS) include neurological and immunological dysfunctions that are linked to cell reduction in these systems. Neural (NSC) and hematopoietic stem cells (HSC) may be targets for the cytotoxic effects of EtOH. Furthermore, protein kinase C (PKC) signal transduction systems of these stem cells may be involved in EtOH-induced cell death. Purified CD34+ human fetal liver hematopoietic stem cells (HSC) and CD133+/nestin+ human neural stem cells (NSC) were exposed to 0.1-10 mM EtOH. A range of indices of cell damage indicated that these doses of EtOH were deleterious to NSC, but had no observable effects on HSC. Furthermore, the colony-forming ability of NSC was completely inhibited by 5 mM EtOH treatment, whereas HSC were unaffected by even 20 mM EtOH. These results suggest that NSC are much more sensitive to EtOH than HSC. Classic and novel PKC isozyme protein expressions in the membrane fraction of cells were differentially affected by EtOH exposure across the two stem cell types. Concentrations of EtOH capable of inducing NSC, but not HSC, death also changed apoptosis-associated PKC isozyme expression in the membrane of NSC, but not HSC. Therefore, PKC expression may mediate the susceptibility of NSC to EtOH-induced cytotoxicity via cell signal transduction pathways. The toxic effect of EtOH on NSC may lead to the decreased neural cell number observed in FAS patients. The comparable immunity of HSC to the deleterious effects of EtOH exposure indicates that the susceptibility of NSC is not simply due to their being stem cells and also may explain the relative lack of hematopoietic problems associated with FAS.

Nicotine and its withdrawal modify dorsal raphe 8-hydroxy-2-(di-n-propylamino) tetralin feeding.

Nicotine (NIC) and its withdrawal modify dorsal raphe (DR) serotonin (5-HT) neurotransmission in ways that may contribute to the body weight loss vs. gain associated with cigarette smoking vs. cessation, respectively. Modifications in feeding to DR infusions of the 5-HT-1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), were used to characterize these potential relationships in the DR-5-HT system during NIC administration vs. withdrawal. Two groups of female rats (total N=45) were implanted for 14 days with subcutaneous Alzet minipumps containing NIC (6 mg/kg/day) or saline. Mid-light cycle (1300-1500 h) 8-OH-DPAT feeding tests occurred three times: (1) 2 days after pump implant, (2) 12 days after pump implant, and (3) 2 days after pump removal. Each feeding test consisted of a 1-h measure of pre-feeding, then a 1-h measure of feeding after DR injection of 8-OH-DPAT (0.6 nmol) or 0.4 microl saline. NIC administration produced acute hypophagia, weight loss, and attenuated 8-OH-DPAT-induced feeding. NIC withdrawal produced acute hyperphagia, weight gain, and a transient increase in 8-OH-DPAT feeding. These findings provide behavioral evidence that systemic NIC modifies the DR 5-HT system in ways that may contribute to NIC's ability to alter feeding and body weight.

Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro.

In some rodent models, there is evidence that hematopoietic stem cells (HSC) can differentiate into neural cells. However, it is not known whether humans share this potential, and, if so, what conditions are sufficient for this transdifferentiation to occur. We addressed this question by assessing the ability of fetal human liver CD34(+)/CD133(+)/CD3(-) hematopoietic stem cells to generate neural cells and astrocytes in culture. We cultured fetal liver-derived hematopoietic stem cells in human astrocyte culture-conditioned medium or using a method wherein growing human astrocytes were separated from cultured, nonadherent hematopoietic stem cells by a semipermeable membrane in a double-chamber co-culture system. Hematopoietic stem cell cultures were probed for neural progenitor cell marker expression (nestin and bone morphogenic protein-2 [BMP-2]) during growth in both culture conditions. RT-PCR, western blotting, and immunocytochemistry assays showed that cells cultured in either condition expressed nestin mRNA and protein and BMP-2 mRNA. HSC similarly cultured in nonconditioned medium or in the absence of astrocytes did not express either marker. Cells expressing these neural markers were transferred and cultured on poly-D-lysine-coated dishes with nonconditioned growth medium for further study. Immunocytochemistry demonstrated that these cells differentiated into astrocytes after 8 days in culture as indicated by their morphology and expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and S100, as well as by their rate of proliferation, which was identical to that of freshly isolated fetal brain astrocytes. These findings demonstrate that neural precursor gene expression can be induced when human hematopoietic stem cells are exposed to a suitable microenvironment. Furthermore, the neural stem cells generated in this environment can then differentiate into astrocytes. Therefore, human hematopoietic stem cells may be an alternative resource for generation of neural stem cells for therapy of central nervous system defects resulting from disease or trauma.