Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord.

To validate human neural precursor cells (NPCs) as potential donor cells for transplantation therapy after spinal cord injury (SCI), we investigated the effect of NPCs, transplanted as neurospheres, in two different rat SCI models. Human spinal cord-derived NPCs (SC-NPCs) transplanted 9 days after spinal contusion injury enhanced hindlimb recovery, assessed by the BBB locomotor test. In spinal compression injuries, SC-NPCs transplanted immediately or after 1 week, but not 7 weeks after injury, significantly improved hindlimb recovery compared to controls. We could not detect signs of mechanical allodynia in transplanted rats. Four months after transplantation, we found more human cells in the host spinal cord than were transplanted, irrespective of the time of transplantation. There was no focal tumor growth. In all groups the vast majority of NPCs differentiated into astrocytes. Importantly, the number of surviving rat spinal cord neurons was highest in groups transplanted acutely and subacutely, which also showed the best hindlimb function. This suggests that transplanted SC-NPCs improve the functional outcome by a neuroprotective effect. We conclude that SC-NPCs reliably enhance the functional outcome after SCI if transplanted acutely or subacutely, without causing allodynia. This therapeutic effect is mainly the consequence of a neuroprotective effect of the SC-NPCs.

Reconsidering pluripotency tests: do we still need teratoma assays?

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

Human neural precursor cells continue to proliferate and exhibit low cell death after transplantation to the injured rat spinal cord.

During the last decade, the interest in stem and progenitor cells, and their applications in spinal cord injuries have steadily increased. However, little is known about proliferation and cell death mechanisms in these cells after transplantation to the spinal cord. The aim of the present project was to study cell turn-over, i.e. total cell number, with time course of proliferation and cell death, in human neural precursor cells (NPCs) after transplantation to the injured rat spinal cord. Immunodeficient rats were subjected to lateral clip compression injuries, transplanted with neurospheres of human forebrain-derived NPCs two weeks after lesion, and sacrificed after 6 h, 1, 3, 10, or 21 days. Cell death was assessed by quantifying human cells immunoreactive for active caspase-3 and calpain 1-dependent fodrin breakdown products (FBDP). The results showed that after an initial drop, the number of implanted cells increased over time after transplantation. Cell proliferation was substantial, with 34% of human cells being immunoreactive for proliferating cell nuclear antigen at 6 h, but which declined over the next few days. The fractions of caspase-3-, and FBDP-immunoreactive cells were remarkably low, together representing 18% of all human cells at 6 h, and rapidly decreasing the next few days. Our results show that already 10 days after spinal cord transplantation of human NPCs as intact neurospheres, the number of human cells exceeded the initially implanted, which was the result of marked cell proliferation in combination with a low rate of apoptotic and non-apoptotic cell death taking place early after transplantation.

Prenatal glucocorticoid exposure affects learning and vulnerability of cholinergic neurons.

Prenatal treatment with synthetic glucocorticoids is commonly used as a treatment for women at risk of preterm delivery. However, little is known about the life-long consequences of these treatments on the fetus. In the present study, we evaluated cognitive function as well as susceptibility of cholinergic neurons to (192)IgG-saporin immunolesion in adult rats after prenatal glucocorticoid treatment. Morris water maze results revealed a significant difference in learning and memory function in adult rats that were prenatally exposed to dexamethasone, and further cognitive deficits after (192)IgG-saporin exposure. Choline acetyl transferase activity was decreased in the cortex of dexamethasone-treated rats compared with controls. In addition, rats prenatally exposed to either dexa, or betamethasone revealed a dramatic decrease in choline acetyl transferase activity compared to control rats after (192)IgG-saporin lesion. We report behavioral and biochemical evidence for altered cognitive function and increased susceptibility of cholinergic neurons to (192)IgG-saporin in adult rats after prenatal glucocorticoid treatment. Taken together, these results suggest that prenatal treatment with dexamethasone could affect cognitive functions and render cholinergic neurons more vulnerable to challenges later in life.

Cell death induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in AtT-20 pituitary cells.

The environmental man-made pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has profound and deleterious effects on the endocrine system, and the pituitary gland is among TCDD endocrine target organs. In the present study, we have investigated the effects of TCDD (1 pM, 0.1 nM and 1 nM) on the AtT-20 pituitary cell line. TCDD induces cell death, with morphological and biochemical changes indicating the occurrence of both apoptotic and necrotic cell death. Exposed cells exhibited apoptotic features including DNA condensation, activation of caspase-3 and exposure of phosphatidylserine (PS) on the outer plasma membrane. Concomitantly, cells with necrotic morphology such as cell swelling and plasma membrane damage were also present. The relative level of Fas ligand mRNA was increased after TCDD exposure, as well as Fas and Fas ligand protein levels detected by Western blotting and immunocytochemistry. Taken together, the results suggest that TCDD induces both necrosis and apoptosis in the pituitary AtT-20 cells and that the Fas/FasL system plays a critical role in inducing necrotic cell death rather than apoptosis (supported by the Swedish Research Council).

Differential regulation of the mitochondrial and death receptor pathways in neural stem cells.

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine-derived multipotent C17.2 NSCs, that cells undergo mitochondria-mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase-9 and -3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase-8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal-regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

Ultrastructural characterization of dissociated embryonic ventral mesencephalic tissue treated with neuroprotectants.

Poor survival and differentiation of grafted dopamine neurons limits the application of clinical transplantation in Parkinson's disease. The survival of grafted dopamine neurons is only improved by a factor of 2-3 by adding neuroprotectants during tissue preparation. We used dye exclusion cell viability and electron microscopy to investigate the effects of the caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural changes in dissociated embryonic mesencephalic cells. In addition, we examined whether the neuroprotectants selectively counteracted specific signs of neurodegeneration. Cell viability decreased significantly over time in both control and treated cell suspensions, but the number of viable cells remaining was significantly higher in tirilazad mesylate-treated cell suspensions. In control samples, the proportion of cells with an ultrastructure consistent with healthy cells decreased from 70%, immediately after dissociation, to 30% after 8 h of incubation. Similar changes were also observed in cell suspensions treated with neuroprotectants. Thus, the neuroprotectants examined did not block the development of specific morphological signs of neurodegeneration. However, when also taking into account that dead cells lysed and disappeared from each cell suspension with time, we found that the total number of remaining viable cells with healthy nuclear chromatin or intact membrane integrity was significantly higher in the tirilazad mesylate-treated group. The results indicate that tirilazad mesylate protects only a small subpopulation of embryonic mesencephalic cells from degeneration induced by mechanical trauma during tissue dissection and dissociation.

Comparison between survival of lazaroid-treated embryonic nigral neurons in cell suspensions, cultures and transplants.

Death of transplanted dopaminergic neurons is induced both during preparation of donor tissue and after intrastriatal grafting. Oxidative stress is thought to be partly responsible for this cell death. In the present study we compared the effects of three lipid peroxidation inhibitors, the lazaroids Tirilazad mesylate, U-83836E and U-101033, on survival of embryonic mesencephalic neurons in different paradigms. The lazaroids were equally potent in preventing serum deprivation-induced death of cultured dopaminergic neurons. In a second set of experiments, mesencephalic suspensions were pretreated with lazaroids and cell survival was analyzed immediately after dissociation, after 2 or 24 h in culture or after intrastriatal transplantation. Lazaroid pretreatment failed to protect mesencephalic neurons in the in vitro paradigms and U-101033E did not protect grafted dopaminergic neurons in contrast to the neuroprotective effects previously reported for U-83836E and Tirilazad. Pretreatment with the iron chelator deferoxamine mesylate did not protect cultured or grafted dopaminergic neurons, nor did it improve neuronal survival in the serum deprivation model. U-83836E and U-101033E, but not Tirilazad, prevented cell death induced by the pro-oxidant tert-butyl hydroperoxide in suspensions. In a final experiment, we found that systemic treatment of the graft recipient rat with Tirilazad mesylate (before and during the first 3 days after grafting) improved survival of transplanted dopaminergic neurons to 180% of control values. Our results show that systemic treatment with a lipid peroxidation inhibitor for 3 days can promote graft survival, but also highlights the poor correlation between neuroprotective effect of pharmacological compounds in vitro and in grafts.