Itzhak Fischer

Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: Disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype

Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron‐like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [ 2002 ] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time‐lapse studies. Similar neurite‐like processes were induced by treating MSC with DMSO only or with actin filament‐depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages.

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot-lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration

Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.

Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury.

Transplants of fibroblasts genetically modified to express BDNF (Fb/BDNF) have been shown to promote regeneration of rubrospinal axons and recovery of forelimb function when placed acutely into the injured cervical spinal cord of adult rats. Here we investigated whether Fb/BDNF cells could stimulate supraspinal axon regeneration and recovery after chronic (4 week) injury. Adult female Sprague-Dawley rats received a complete unilateral hemisection injury at the third cervical spinal cord segment (C3). Four-five weeks later the injury site was exposed and rats received transplants of unmodified fibroblasts (Fb/UM) or Fb/BDNF. Four-five weeks after transplantation, locomotor recovery was examined on a test of forelimb usage and regeneration of supraspinal axons was studied following injection of the anterograde tracer biotin dextran amine (BDA). Rubrospinal tract (RST), reticulospinal tract (ReST), and vestibulospinal tract (VST) axons regenerated into transplants of either Fb/UM or Fb/BDNF but the length of axonal growth was significantly different in the two groups. The absolute distance of ReST growth was 1.8-fold greater in Fb/BDNF than in Fb/UM and the absolute distance of growth of RST and VST axons showed a statistically significant 4-fold increase. All three types of regenerated axons occupied a greater proportional length of Fb/BDNF transplants than of Fb/UM transplants. Only VST axons extended into the host spinal cord caudal to the Fb/BDNF grafts, but these axons were sparse. Rats receiving Fb/BDNF used both forelimbs together to explore walls of a cylinder more often than rats receiving Fb/UM, indicating partial recovery of forelimb usage. These results demonstrate that fibroblasts genetically modified to express BDNF promote axon regeneration from supraspinal neurons in the chronically injured spinal cord with accompanying partial recovery of locomotor performance.

THE LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN IS AN IG SUPERFAMILY MEMBER THAT MEDIATES SELECTIVE NEURONAL GROWTH AND AXON TARGETING

The formation of brain circuits requires molecular recognition between functionally related neurons. We report the cloning of a molecule that participates in these interactions. The limbic system-associated membrane protein (LAMP) is an immunoglobulin (Ig) superfamily member with 3 Ig domains and a glycosyl-phosphatidylinositol anchor. In the developing forebrain, lamp is expressed mostly by neurons comprising limbic-associated cortical and subcortical regions that function in cognition, emotion, memory, and learning. The unique distribution of LAMP reflects its functional specificity. LAMP-transfected cells selectively facilitate neurite outgrowth of primary limbic neurons. Most striking, administration of anti-LAMP in vivo results in abnormal growth of the mossy fiber projection from developing granule neurons in the dentate gyrus of the hippocampal formation, suggesting that LAMP is essential for proper targeting of this pathway. Rather than being a general guidance cue, LAMP likely serves as a recognition molecule for the formation of limbic connections.

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Transplantation of Neuronal and Glial Restricted Precursors into Contused Spinal Cord Improves Bladder and Motor Functions, Decreases Thermal Hypersensitivity, and Modifies Intraspinal Circuitry

Transplanting neuronal and glial restricted precursors (NRP/GRP) into a midthoracic injury 9 d after contusion improved bladder and motor function, diminished thermal hypersensitivity, and modified lumbosacral circuitry compared with operated controls (OP-controls). Histological analysis showed that NRP/GRP survived, filled the lesion site, differentiated into neurons and glia, and migrated selectively. Volume of spinal cord spared was increased in NRP/GRP recipients, suggesting local protection. Bladder areflexia developed in both operated groups, but NRP/GRP recipients exhibited an accelerated recovery, with decreased micturition pressure and fewer episodes of detrusor hyperreflexia. Because noradrenergic receptors proliferate after spinal injury and descending noradrenergic pathways contribute to regulation of bladder control, we examined the effects of administering an α-1A-adrenergic antagonist, Tamsulosin, on urodynamics. This improved all cystometric parameters in both operated groups, and micturition pressure in NRP/GRP rats recovered to normal levels. Both operated groups initially showed increased sensitivity to a thermal stimulus applied to the tail; the NRP/GRP rats showed significant improvement over time. NRP/GRP grafts also produced greater recovery of hindlimb function in several tests, although both groups showed persistent and similar deficits in locomotion on a grid. Because bladder, hindlimb, and tail sensory and motor functions are organized through lumbosacral cord, we examined descending and primary afferent projections at L6-S1. The density of serotonergic, noradrenergic, and corticotrophin releasing factor-positive fibers increased in the NRP/GRP group compared with OP-controls, suggesting some sparing and/or sprouting of these modulatory pathways. Immunocytochemical staining density of dorsal root axons in the dorsal horn increased in the OP-controls but appeared normal in the NRP/GRP group. Synaptophysin immunoreactivity in the lumbosacral dorsal horn was similar among groups, consistent with restoration of synaptic density in both groups of operated animals but by different pathways. We suggest that local protection provided by NRP/GRP resulted in increased sparing/sprouting of descending pathways, which prevented sprouting by dorsal root axons, and that this modification in lumbosacral circuitry contributes to the recovery of function.

Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord.

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.

EFFECTS OF PLATING DENSITY AND CULTURE TIME ON BONE MARROW STROMAL CELL CHARACTERISTICS

OBJECTIVE Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. MATERIALS AND METHODS Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations. RESULTS We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. CONCLUSION Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.

Grafted Neural Progenitors Integrate and Restore Synaptic Connectivity across the Injured Spinal Cord

Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost after spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. One week later, BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immunoelectron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.