PURPOSE OF REVIEW Central to the obstacles to be overcome in moving promising cell-based therapies from the laboratory to the clinic is that of determining which of the many cell types being examined are optimal for repairing particular lesions. RECENT FINDINGS Our studies on astrocyte replacement therapies demonstrate clearly that some cells are far better than others at promoting recovery in spinal cord injury and that, at least in some cases, transplanting undifferentiated precursor cells is far less useful than transplanting specific astrocytes derived from those precursor cells. But further comparison between different approaches is hindered by the difficulties in replicating results between laboratories, even for well defined pharmacological agents and bioactive proteins. These difficulties in replication appear most likely to be due to unrecognized nuances in lesion characteristics and in the details of delivery of therapies. SUMMARY We propose that the challenge of reproducibility provides a critical opportunity for refining cell-based therapies. If the utility of a particular approach is so restricted that even small changes in lesions or treatment protocols eliminate benefit, then the variability inherent in clinical injuries will frustrate translation. In contrast, rising to this challenge may enable discovery of refinements needed to confer the robustness needed for successful clinical trials.

PURPOSE: Some patients with intractable metabolic stone disease experience narcotic dependence, which cannot be managed with standard treatments. We offered these patients renal autotransplantation with a modified pyelovesicostomy as an alternative solution. MATERIALS AND METHODS: Renal autotransplantation with pyelovesicostomy was performed for 15 kidneys in 12 patients (3 bilateral, 2 solitary), 9 female and 3 male, with a mean age of 33.8 years (range 16 to 55). The etiology of metabolic stone disease was calcium oxalate (40%), cystinuria (33%), type 1 renal tubular acidosis (14%), calcium oxalate/urate (7%) and medullary sponge kidney (7%). Patients reported that lifetime stone events ranged from 10 to more than 70, that underwent an average of 3 to 4 surgical interventions per year in the previous 2 years and that they were dependent on daily oral narcotics for stone related pain. RESULTS: All 15 kidneys were successfully autotransplanted with a mean followup of 41.8 months (range 3 to 74). We used a modified pyelovesicostomy with ureteral strip in 13 and standard Boari tube in 2 cases. All patients continued to pass small stone debris per urethra with minimal symptoms. Of 12 patients 11 (92%) were weaned off daily narcotics. There have been 17 stone episodes in 4 patients (3 cystinuria) for which medical intervention and pain medication was required. The number of urological procedures/patients before (155/12 [12.9]) and after (8/12 [0.66]) autotransplantation was dramatically reduced (paired t test p = 0.0001). The preoperative mean estimated glomerular filtration rate was 77.2 cc/minute, and 73.5, 71.9, 79.2 cc/minute at 12, 36 and 60 months, respectively. CONCLUSIONS: Renal autotransplantation and pyelovesicostomy offer patients with intractable metabolic stone disease the opportunity to improve quality of life and to decrease daily narcotic use.

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.

ABSTRACT: Chemotherapy in cancer patients can be associated with serious short and long-term neurological adverse effects, such as leukoencephalopathy and cognitive impairment. The underlying cellular basis for these adverse effects is poorly understood. We have found that three mainstream chemotherapeutic agents - BCNU (carmustine), cisplatin and cytarabine (cytosine arabinoside)- applied at clinically relevant exposure levels are even more toxic for progenitor cells of the CNS and non-dividing oligodendrocytes than they are for multiple cancer cell lines. Moreover, at sub-lethal doses these agents compromise the capacity of progenitor cells for continued cell division. When administered systemically, these chemotherapeutic agents were associated with increased cell death and decreased cell division in several regions of the CNS, often with effects that lasted for weeks after drug administration. These findings provide a potential cellular basis for understanding adverse neurological consequences following chemotherapy, and thus identifying targets for preventing or alleviating such damage.

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells--a precursor cell population restricted to oligodendrocyte and astrocyte lineages--offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.

We have found that the tripotential glial-restricted precursor (GRP) cell of the embryonic rat spinal cord can give rise in vitro to bipotential cells that express defining characteristics of oligodendrocyte-type-2 astrocyte progenitor cells (O2A/OPCs). Generation of O2A/OPCs is regulated by environmental signals and is promoted by platelet-derived growth factor (PDGF), thyroid hormone (TH) and astrocyte-conditioned medium. In contrast to multiple observations indicating that oligodendrocyte precursor cells in the embryonic day 14 (E14) spinal cord are ventrally restricted, GRP cells are already present in both the dorsal and ventral spinal cord at E13.5. Ventral-derived GRP cells, however, were more likely to generate O2A/OPCs and/or oligodendrocytes than were their dorsal counterparts when exposed to TH, PDGF, or even bone morphogenetic protein-4. The simplest explanation of our results is that oligodendrocyte generation occurs as a result of generation of GRP cells from totipotent neuroepithelial stem cells, of O2A/OPCs from GRP cells and, finally, of oligodendrocytes from O2A/OPCs. In this respect, the responsiveness of GRP cells to modulators of this process may represent a central control point in the initiation of this critical developmental sequence. Our findings provide an integration between the earliest known glial precursors and the well-studied O2A/OPCs while opening up new questions concerning the intricate spatial and temporal regulation of precursor cell differentiation in the CNS.

One of the most extensively studied of mammalian cells is the oligodendrocyte, the myelin-forming cell of the central nervous system. The ancestry and development of this cell have been studied with every approach utilized by developmental biologists. Such detailed efforts have the potential of providing paradigms of relevance to those interested in analyzing the ancestry and development of any cell type.One of the striking features of studies on the development of oligodendrocytes is that different analytical approaches have led to strikingly different theoretical views regarding the ancestry of these cells. On one extreme is the hypothesis that the steps leading to the generation of oligodendrocytes begin with the generation of a glial-restricted precursor (GRP) cell from neuroepithelial stem cells. GRP cells are thought to be capable of giving rise to all glial cells (including oligodendrocytes and multiple astrocyte populations), but not to neurons, a process that appears to require progression through further stages of greater lineage restriction. On the other extreme is the hypothesis that oligodendrocytes are derived from a precursor cell that generates only motor neurons and oligodendrocytes, with astrocytes being generated through a separate lineage.In this review, we critically consider the various contributions to understanding the ancestry of oligodendrocytes, with particular attention to the respective merits of the GRP cell vs. the motor neuron-oligodendrocyte precursor (MNOP) cell hypothesis. We draw the conclusion that, at present, the strengths of the GRP cell hypothesis outweigh those of the MNOP hypothesis and other hypotheses suggesting oligodendrocytes are developmentally more related to motor neurons than to astrocytes. Moreover, it is clear from existing data that, following the period of motor neuron generation, the major glial precursor cell in the embryonic spinal cord is the GRP cell, and that multiple previous studies on the earliest stages of oligodendrocyte generation in the developing spinal cord have been focused on a differentiation stage of GRP cells.

ABSTRACT : BACKGROUND : Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. RESULTS : Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. CONCLUSION : Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.

In our attempts to understand how the balance between self-renewal and differentiation is regulated in dividing precursor cells, we have discovered that intracellular redox state appears to be a critical modulator of this balance in oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. The intracellular redox state of freshly isolated progenitor cells allows prospective isolation of cells with different self-renewal characteristics, which can be further modulated in opposite directions by prooxidants and antioxidants. Redox state is itself modulated by cell-extrinsic signaling molecules that alter the balance between self-renewal and differentiation: growth factors that promote self-renewal cause progenitors to become more reduced, while exposure to signaling molecules that promote differentiation causes progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell-extrinsic signaling molecules antagonize their effects on self-renewal and differentiation, further suggesting that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism. A further example of the potential relevance of intracellular redox state to development processes emerges from our attempts to understand why different central nervous system (CNS) regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of O-2A progenitor cells (O-2A/OPCs) isolated from different regions indicates that these developmental patterns are consistent with properties of the specific O-2A/OPCs resident in each region. Marked differences were seen in self-renewal and differentiation characteristics of O-2A/OPCs isolated from cortex, optic nerve, and optic chiasm. In conditions where optic nerve-derived O-2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm-derived cells after 5 days and from cortical O-2A/OPCs after only 7-10 days. These differences, which appear to be cell intrinsic, were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve-, chiasm-, and cortex-derived O-2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well-characterized inducers of oligodendrocyte generation, was inversely related to the extent of self-renewal observed in basal division conditions. These results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions. Strikingly, O-2A/OPCs isolated from cortex and analyzed immediately upon isolation were more reduced in their redox state than were optic nerve-derived cells, precisely as would be predicted from our analysis of the role of redox state in modulating the balance between self-renewal and differentiation. Chiasm-derived cells, which exhibited self-renewal properties intermediate between cortex- and optic nerve-derived cells, were more reduced than optic nerve cells but more oxidized that cortical O-2A/OPCs.

ABSTRACT: BACKGROUND: Cancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the body's own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem. RESULTS: We found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment. CONCLUSIONS: Our studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

Different CNS regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of oligodendrocyte-type-2 astrocyte progenitor cells (here abbreviated as O-2A/OPCs) isolated from different regions indicates these developmental patterns are consistent with properties of the specific O-2A/OPCs resident in each region. Marked differences were seen in self-renewal and differentiation characteristics of O-2A/OPCs isolated from cortex, optic nerve and optic chiasm. In conditions where optic nerve-derived O-2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm-derived cells after 5 days and from cortical O-2A/OPCs only after 7-10 days. These differences, which appear to be cell-intrinsic (and may be related to intracellular redox state), were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve-, chiasm- and cortex-derived O-2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well-characterized inducers of oligodendrocyte generation, was inversely related to the extent of self-renewal observed in basal division conditions. Our results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes, and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions.