Rat Bone Marrow Progenitor Cells Transduced In Situ by rSV40 Vectors Differentia - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Jean-Pierre Louboutina, Bianling Liua, Beverly A.S. Reyesb, Elisabeth J. Van Bockstaeleb, David S. Strayera作者单位：aDepartment of Pathology, Anatomy, and Cell Biology andbDepartment of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, Pennsylvania, USA ) @" }+ T1 h  s8 }: @
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      【摘要】! i+ j  R# n1 U: O* {. a
   Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0¨C16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG  throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG  cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells.
      【关键词】 Stem cells Bone marrow Gene transfer SV Brain Hippocampus
   INTRODUCTION
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Considering the characteristics of the vectors used so far, gene transfer in the brain has been limited to targeting mature neuronal cells only, not precursor cells. However, the stability of gene transfer could be compromised if partially damaged cells are transduced, as is likely in several neurodegenerative diseases (e.g., Alzheimer and Parkinson diseases). Therefore, an approach based on somatic gene transfer to postmitotic cells might not result in permanent correction. Recently, a rather limited population of brain progenitor cells was described mainly in two areas of the brain, the dentate gyrus (DG) of the hippocampus and the periventricular subependymal zone. These groups of cells could participate not only in neurogenesis throughout life but also in repair of brain lesions. One might then genetically engineer brain stem cells in situ so that their proliferation after injury of the brain would lead to functional brain cells expressing the transgene. However, the number of progenitor cells is very limited in the adult brain, and adult stem cells may have a shorter life span and different properties than pluripotent stem cells.

It has been shown that some bone marrow (BM) stem cells were able to migrate to the brain and differentiate into different types of brain cells, in rodents , as well as for gene delivery. However, BM transplantation can lead to serious immune side effects, such as graft-versus-host disease.

One solution would be to transduce HSCs directly in the BM. However, despite the potential promise of such an approach, in situ gene transfer to hematopoietic stem/progenitor cells by intramarrow injection of viral vectors has been rarely reported, and the distribution of transgene-expressing cells in the body after intramarrow gene delivery has never been described. In a previous study , we reported successful transduction of the femoral BM of rats by intramarrow injection of a T-antigen (Tag)-deleted recombinant SV40 vector carrying a marker gene (FLAG epitope appended to HIV-1 Nef as a carrier protein). Control rats received a control rSV40 vector. Transgene expression in the blood endured throughout the 16 months of the study: 4%¨C12% (average, 5%) of blood nucleated cells of all lineages were positive for FLAG through the end of the study, as well as 25% of femoral marrow cells. Circulating peripheral blood mononuclear cells and granulocytes expressed the transgene. However, the putative migration of HSCs to the brain under these experimental conditions had not been examined yet. Because it has been demonstrated that brain progenitor cells were mainly located in the DG and in the periventricular subependymal zone, we focused the present study on the distribution and phenotype of FLAG-positive cells in these areas at different times after injection of SV40(Nef-FLAG) into the BM.5 _* a/ ]. U+ B( _6 Y$ [

MATERIALS AND METHODS

Animals

Female Sprague-Dawley rats (200¨C250 g) were purchased from Charles River Laboratories (Wilmington, MA, http://www.criver.com). Protocols for injecting and sacrificing animals were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee and are consistent with Association for Assessment and Accreditation of Laboratory Animal Care standards.6 h: V# n" q5 Q

Vector Production

The general principles for making recombinant replication-defective SV40 viral vectors have been previously reported . rSV40 viral vectors, carrying cDNA encoding HIV Nef protein with a carboxyl-terminal FLAG epitope tag with a cytomegalovirus immediate early promoter were used. Infectious titers of such rSV40 viral vectors are generally 1011¨C1012 infectious units (IU)/ml.

Procedure of Percutaneous Intrafemoral Bone Marrow Injection
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Sprague-Dawley rats were anesthetized, and the region from the hip to the knee joint was shaved. The knee was flexed to 90¡ã, a 271/2G-gauge needle attached to a 1-ml syringe was lodged between the condyles at the top of the femur, and access to the BM cavity was gained by applying gentle twisting and pressure. SV(Nef-FLAG) viral vector (1 x 1011 IU) in 100 µl of phosphate-buffered saline (PBS) was injected into bone marrow cavity of each femur in test rats. In the same manner, control rats (n = 3) received 1 x 1011 IU of SV(BUGT) viral vector in 100 µl of PBS. Peripheral blood cells (average, 5%) and femoral marrow cells (average, 25%) were FLAG-positive in all test rats throughout the study, whereas no peripheral blood cells or femoral marrow cells were positive for FLAG in the control rats.

Sample Processing, o6 {( E2 k* e; |: H3 b
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After a survival period of 1, 4, or 16 months (n = 3, 2, and 3, respectively), rats were anesthetized via intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL, http://www.abbott.com) at 60 mg/kg and perfused transcardially although the ascending aorta with 10 ml of heparinized saline and 1,000 ml of ice-cold 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, www.emsdiasum.com) in 0.1 M phosphate buffer (pH 7.4). Immediately following perfusion-fixation, the rat brains were dissected out, placed in 4% paraformaldehyde for 24 hours and then in a 30% sucrose solution for 24 hours, and then frozen in methylbutane cooled in liquid nitrogen. The samples were cut on a cryostat (10-µm sections).

Immunocytochemistry
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For immunofluorescence, the coronal cryostat sections (10 µm thick) were processed for immunocytochemistry with an indirect immunofluorescence technique. Blocking was performed by a 60-minute incubation with 10% goat or 10% donkey serum in 0.10 M PBS (pH 7.4). Then, cryostat sections were incubated with antibodies at dilutions described earlier. Incubation with primary antibody was performed for 1 hour and followed by incubation for 1 hour with a secondary antibody diluted 1:100. Incubations were performed at room temperature. Double immunofluorescence was performed as previously described . Each incubation was followed by extensive washing with PBS. To stain the nuclei, the mounting medium contained 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Specimens were finally examined under a Leica DMRBE microscope (Leica, Wetzlar, Germany, http://www.leica.com). Negative controls consisted of preincubation with PBS and 0.1% bovine serum albumin, substitution of nonimmune isotype-matched control antibody for primary antibody, and/or omission of the primary antibody. The 1-month, 4-month, and 16-month sections were immunostained on the same day and at the same time as the controls. The experiments were repeated several times (more than four times, on average).% w7 u$ ^: r9 T* T

Antibodies

Different primary antibodies were used: mouse and rabbit anti-FLAG (1:100), rabbit anti-glial fibrillary acidic protein (anti-GFAP) antibodies (1:100) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), rabbit anti-GFAP (1:200) (Immunostar, Inc., Hudson, WI, http://www.immunostar.com), mouse anti-NeuN (1:100) (Chemicon, Temecula, CA, http://www.chemicon.com), mouse anti-rat CD68/ED1 (1:50) (Serotec Ltd., Oxford, U.K., http://www.serotec.com), mouse anti-OX-42 (rat CD11b) (1:50) (Accurate Chemical and Scientific Corporation, Westbury, NY, http://www.accuratechemical.com), and mouse anti-proliferating cell nuclear antigen (anti-PCNA) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). The following secondary antibodies were used at a 1:100 dilution: fluorescein isothiocyanate (FITC)-conjugated and tetramethylrhodamine B isothiocyanate (TRITC)-conjugated goat anti-mouse, TRITC-conjugated goat anti-rabbit, FITC-conjugated sheep anti-rabbit (Sigma-Aldrich), and FITC- and TRITC-conjugated donkey anti-mouse (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) antibodies.. F6 u+ [' q. O5 i, `) f$ {5 L

Staining of Neurons Using NeuroTrace' |% E# E: D+ L- j# _
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After rehydration in 0.1 M PBS, pH 7.2, sections were permeabilized in PBS plus 0.1% Triton X-100 for 10 minutes, washed twice for 5 minutes in PBS, and then stained by NeuroTrace (NT, 1:100) (Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com), a fluorescent Nissl stain, for 20 minutes at room temperature. The sections were washed in PBS plus 0.1% Triton X-100 and then two times with PBS and then let stand for 2 hours at room temperature in PBS before being counterstained with DAPI. Combination NeuroTrace antibody staining was performed using the primary and secondary antibodies staining first, followed by staining with the fluorescent Nissl stain. As described above, incubation with the primary antibody was performed for 1 hour and followed by incubation for 1 hour with a secondary antibody diluted 1:100. Each incubation was followed by extensive washing with PBS. To stain the nuclei, the mounting medium contained DAPI (Vector Laboratories). The experiments were repeated three times and were done the same day for the different sections considered.
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General Morphology
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General morphology of the brain was assessed by neutral red staining performed on cryostat sections to determine the level of the section before immunocytochemistry., L9 q& [- G; A0 n4 P( P

Morphometry

Transduction was assessed for each brain by serial cryosectioning (10-µm coronal sections) and immunostaining for FLAG. FLAG-positive cells were counted on serial sections (at least five different sections) of the rat brain, focusing on the DG and its different components. The percentage of the different cell types positive for FLAG was assessed after double immunostaining of FLAG and the cell marker.' Z* C# ~' z  D- E+ }3 q, Y

RESULTS7 U) @: z, F$ Z7 j; Y4 O! t: m

Transgene Expression in Cells with Neuronal Phenotype in the Dentate Gyrus* I5 S# p& m3 ]0 ]/ G% V) @" A

We focused the study on the DG, which is a part of the hippocampus composed of the granule cell layer (GCL), which is formed of inner (upper) and outer (lower) blades surrounding the hilus area. Sixteen months after injection of SV(Nef-FLAG) directly into the BM, FLAG expression was observed in the brain in some cells with neuronal morphology (Fig. 1A). No FLAG expression was seen in the DG in control animals either injected with saline or injected with a control SV, or when the primary antibody was replaced by a nonimmune isotype-matched immunoglobulin (Fig. 1A). Very rare FLAG-expressing cells were detected in the DG 4 months after injection of the vector into the BM. No FLAG-expressing cells were observed in the DG 1 month after injection of SV(Nef-FLAG) into the BM. Some of the FLAG-expressing cells coimmunostained with NeuN, a neuronal marker, in the GCL, as well as in the hilus area (Fig. 1B). The morphology of NeuN-positive FLAG-expressing cells was strongly suggestive of neurons from the DG, with some characteristics of basket and pyramidal cells (Fig. 1B). FLAG-expressing cells that were not NeuN-positive were also observed in the same areas, suggesting that neurons were not the only cell type expressing the transgene (Fig. 2A). Similar results were seen when neurons were stained by NT, a fluorescent Nissl stain; many FLAG-positive cells, but not all, were stained by NT. No FLAG-expressing cells were seen when a control SV was injected into the BM. In this case, there was also no bleed-through between the FITC and the TRITC channels (Fig. 2B). Figure 3 shows the distribution of cells positive for both FLAG and NeuN in different serial sections of the DG.
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Figure 1. Expression of the transgene FLAG in the dentate gyrus after injection of SV(Nef-FLAG) in the BM. (A): FLAG expression in the dentate gyrus (DG) of rats whose BM has been injected with SV(Nef-FLAG). Cryostat sections of brain immunostained with anti-FLAG antibody (BM-FLAG) showed FLAG-positive cells, often with neuron-like morphology. DG immunostaining for FLAG was negative in control rats where BM had been injected with a control SV40 vector (control-FLAG), and when the primary antibody was omitted (no first antibody). Scale bar = 10 µm. (B): Morphology of DG cells doubly positive for FLAG and NeuN 16 months after injection of SV(Nef-FLAG) into the bone marrow. High magnification photographs show the neuron-like morphology of the DG cells doubly positive for FLAG and NeuN: either basket (A), or pyramidal (B, C) cells. Scale bar = 10 µm (A, B), 5 µm (C). Abbreviation: BM, bone marrow.

Figure 2. Expression of FLAG in cells positive for Neu-N. (A): Cryostat sections of brain from rats whose BM has been injected with SV(Nef-FLAG) 16 months previously were simultaneously immunostained for NeuN and FLAG. The overlay composite highlights the many FLAG-positive cells that also labeled for NeuN (first row, low magnification; second row, high magnification). Control groups are shown in the third row (rats whose BM has been injected with a control SV40 vector) and the fourth row (SV recipient immunostaining when the primary Ab was replaced by a nonimmune isotype-matched Ab). Scale bar = 120 µm (first row), 30 µm (second, third, and fourth rows). (B): Cryostat sections immunostained for FLAG and stained for NT 16 months after the injection of SV(Nef-FLAG) into the bone marrow. Numerous (but not all) cells were immunostained for FLAG and stained for NT (BM-inj). No expression of FLAG was seen when the BM had been injected with a control SV vector (BM-ctl). Scale bar = 120 µm. Abbreviations: Ab, antibody; BM, bone marrow; ctl, control; DAPI, 4,6-diamidino-2-phenylindole; inj, injected; NT, NeuroTrace.

Figure 3. Location of FLAG expression in neurons of the dentate gyrus 16 months after injection of SV(Nef-FLAG) in the bone marrow. Schematic distribution of cells positive for both NeuN and FLAG in the dentate gyrus on four sections spaced 200 µm apart from each other. Each dot represents a cell. Abbreviations: CC, corpus callosum; cing, cingulum; DG, dentate gyrus.. n7 G& L9 D9 i- y4 Y( x4 ]& x+ U
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The percentage of NeuN-positive cells expressing FLAG was measured on several serial cryostat sections (at least five sections) for each sample examined. The inner and outer blades of the GCL were examined, as well as the hilus area. Sixteen months after in situ injection of SV(Nef-FLAG) into the bone marrow, the percentage of NeuN-positive cells expressing FLAG was approximately 2.4% in the different areas examined, whereas there were very few transgene-positive cells 4 months after the injection (Table 1).

Table 1. Number of transgene-positive cells in the NeuN-positive cells of the dentate gyrus after injection of SV(Nef-FLAG) into the rat bone marrow

Transgene Expression in Non-Neuronal Cells in the Dentate Gyrus
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FLAG expression was observed in cells with the morphology of microglial cells (Fig. 4A). Some of the FLAG-expressing cells coimmunostained with antibodies against CD11b-C3bi and CD68, markers of microglial cells (Fig. 4B¨C4D). Very rare GFAP-positive cells were expressing FLAG (Fig. 4E). Morphometric studies showed that 48.6% of the FLAG-expressing cells were NeuN-positive and that 49.7% of the FLAG-positive cells were expressing markers of microglial cells; only 1.6% of FLAG-positive cells were GFAP-positive.8 F" C% g9 h% T/ b! {
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Figure 4. Transduction of bone marrow leads to expression of the transgene in microglial cells. (A): Cryostat sections of different brains were immunostained for FLAG and the microglial cells markers CD11b and CD68/ED1 4 (FLAG-4mo) and 16 (FLAG-16mo) months after the injection of SV(Nef-FLAG) into the bone marrow. FLAG-positive cells showing morphology characteristic of microglial cells (arrows). (B): Some CD11b cells coimmunostained with FLAG. Note that not all FLAG-expressing cells are CD11b-positive. Note neuron-like cells positive for FLAG (arrow). (C): A cell positive for FLAG (arrow) coimmunostained for ED1. (D): Left panel, ED1 immunostaining in the spleen showed numerous positive cells; right panel, no staining for FLAG was seen in the brain of a control rat that had SV(BUGT) injection. (E): Cryostat sections of brain immunostained for FLAG and GFAP 16 months after injection of SV(Nef-FLAG) into the bone marrow. Occasional GFAP-positive cells coimmunostained for FLAG (arrows). The area shown iss the one where the number of GFAP-positive cells coimmunostained for FLAG was the highest in the samples examined. In most of the other areas of the DG, GFAP-positive cells did not coimmunostain for FLAG (not shown). Scale bar = 20 µm (A¨CC), 40 µm (D), and 20 µm (E). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GCL, granule cell layer; GFAP, glial fibrillary acidic protein; mo, months./ y% \8 ~/ O6 d; U; c( ~
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Expression of PCNA in the Dentate Gyrus
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It has been previously shown that proliferating cells can be observed in the DG. Using PCNA as a markers of proliferating cells, we observed rare PCNA-positive cells in the GCL, suggesting, however, that some cells were proliferating in the DG (Fig. 5). Few of the PCNA-positive cells expressed FLAG (Fig. 5), suggesting that only rare transgene-positive cells were proliferating.
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Figure 5. Brain cryostat sections coimmunostained for FLAG and PCNA 16 months after the injection of SV(Nef-FLAG) into the bone marrow. (A): Rare PCNA-positive cells were present in the dentate gyrus. (B): A PCNA-positive cell (1) coimmunostained with FLAG. A FLAG-positive cell (indicated by 2) does not coimmunostain with PCNA. Scale bar = 20 µm (A), 100 µm (B). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GCL, granule cell layer; PCNA, proliferating cell nuclear antigen.
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FLAG Expression in the Periventricular Subependymal Zone
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One and 4 months after the injection of SV(Nef-FLAG) in the BM, there were no or few cells, respectively, positive for the transgene in the periventricular subependymal zone (PSZ), whereas 16 months after the injection of the vector in the BM cavity, numerous FLAG-positive cells were found in the PSZ and, as in the DG, approximately 50% of FLAG-positive cells were stained by NT. There was no expression of FLAG when the primary antibody was replaced with a nonimmune isotype-matched immunoglobulin or when the BM was injected with a control rSV40 vector. Moreover, when the BM has been injected with a control vector, there was no bleed-through between the green (FITC) and the red (TRITC) channels (Fig. 6). The immunostaining studies of the PSZ were repeated three times, and all sections (controls and tests ones) were immunostained simultaneously. These data suggest that FLAG-expressing cells can be found in other areas than the DG.

Figure 6. Expression of FLAG in the periventricular subependymal zone after injection of SV(Nef-FLAG) in the BM. Brain cryostat sections immunostained for FLAG and stained for NT 16 months after the injection of SV(Nef-FLAG) into the bone marrow. Numerous (but not all) cells present around the lateral ventricle were immunostained for FLAG and stained for NT. No expression of FLAG was seen when the primary antibody was replaced by a nonimmune isotype-matched immunoglobulin (no first Ab) or when the BM had been injected with a control SV vector (control). Scale bar = 60 µm. Abbreviations: Ab, antibody; BM, bone marrow; DAPI, 4,6-diamidino-2-phenylindole; inj, injected; NT, NeuroTrace.6 \' O  p! _- e0 y! z/ z

DISCUSSION- c6 ~; I6 S( H- p6 }

In these studies, we demonstrate that gene delivery to the BM results in transgene expression in a mixed population of cells in the brain, particularly in the DG. By administering a gene (FLAG)-carrying SV40-derived vector via intramarrow injection, DG neurons, microglia, and some astrocytes were observed to express the transgene. FLAG expression was not seen within the first few weeks after injection, and minimal expression was seen in brains 4 months postinjection. We interpret these data to indicate that neurogenesis continues in the DG well into adult life and that at least some of the cells of diverse lineages in the DG cells that are generated in this fashion derive from one or more populations of cells that were resident in the BM at the time of injection.
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There are other possible explanations for these observations, but they do not account for all of the data presented. Thus, even though rSV40 vectors transduce central nervous system (CNS) cells effectively when given by direct intracerebral injection .9 H* a7 x+ T- j& J) _& M, a1 a

Other investigators have also reported data that led them to conclude that mature CNS cells may derive from progenitors in the BM. Thus, neural stem cells reside in the PSZ and hippocampus and can give rise to all three types of CNS cells . Because, as with the current report, no stress or injury to the brain was used in those studies, the apparent differentiation of BM progenitors seen is most likely a physiologic phenomenon, unassociated with adaptive or regenerative responses to organ injury.
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There is evidence that the adult rodent brain may attempt to repair itself by neurogenesis after various types of injury .& p: Y. f  z  x4 `2 {8 F
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Whether such activity occurs in humans in physiologic or pathologic states is unclear. Cogle et al.  found that women who received BM transplants from men showed neurons in the hippocampus of donor origin. They observed that the development of these neurons took a long time, which is consistent with our present observations.; I$ t0 ?# @3 A/ N) Y6 k
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Several reports in experimental animals suggest that BM-derived cells may trans-differentiate into CNS cells of one or more lineages. Thus, BM from male mice, transplanted into hematopoietically compromised female recipients, gave rise to Y-chromosome-positive CNS cells that expressed neuronal markers , even if alternative explanations for these observations have not been completely ruled out.
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The mechanism(s) by which such trans-differentiation may occur are not clear. The first step in this process would be homing of the BM-derived population(s) to the brain. Several molecular species have been suggested as being involved in this process, including vascular and extracellular matrix molecules .

The second step in the process would be differentiation into CNS cells. How this occurs is, similarly, unclear .1 {7 e* z  d! c( v, M8 v
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In attempts to improve the effectiveness of gene delivery for expression in the peripheral blood, other groups have used direct injection of viral gene delivery vehicles into the BM, as an alternative to ex vivo transduction and reimplantation .

Examination of the brain, as reported here, and other organs (data not shown) was undertaken to assess the extent to which FLAG expression was detectable outside of the BM. Our finding that multiple lineages of CNS cells expressed FLAG long after the initial injection suggested that these cells were likely to have originated from progenitors in the BM. We found that approximately 5% of DG cells were FLAG-positive. To put this figure into perspective, it should be noted that the femoral marrow is responsible for approximately 10% of total BM hematopoiesis. Considering the manner in which transduction was performed, any cell population in the BM at the time of vector injection might be transduced. Our studies thus do not establish the identity of the BM cell population(s) that lead to transgene expression in CNS: they may be of hematopoietic, stromal, or other origin. Nor, because organ injury was not part of our studies, do our data provide guidance as to the ability of BM-resident progenitor cells to trans-differentiate into other lineages (e.g., hepatocytes or cardiac myocytes) as part of a repair process.' N! V) p2 Z( b

Different studies have been reported either supporting or arguing against the idea that BM cells can trans-differentiate into brain-specific cells. However, the divergent results reported could reflect differences in the methods and models used. Most of the studies that do not support the hypothesis of BM to CNS trans-differentiation used protein products of transgenes as markers  can in part be attributable to short time intervals between treating the animals and harvesting the brains.7 y/ D: ~* o1 b2 W4 A. s' r) ~

It also seems that demonstration of BM-derived cells in the brain surely depends on the experimental system in which the hypothesis is tested. So far, the majority of the studies concerning the fate of HSCs in the brain were based on transplantation of HSCs, which were often treated ex vivo with various growth factors before transplantation into an irradiated recipient. This experimental design may not allow for direct modeling of normal cellular physiology and development. For example, BM cells other than HSCs (stromal cells for example) could play a role in trans-differentiation of BM cells into CNS cells. Treating HSCs with growth factors in vitro may lead some of these cells to differentiate, potentially precluding homing to the CNS or differentiation to CNS cell types. Irradiation, or cytotoxic conditioning, and the need for engraftment may also select for a phenotype in surviving cells and in the environment to which they adapt themselves, and in so doing, select against a trait that is important in trans-differentiation. All these experimental interventions create conditions that are variably removed from the unperturbed physiological situation. To some extent, our experimental system avoids some of these alterations: transducing bone marrow directly leaves the different populations of cells present in the BM undisturbed, and the rSV40 gene delivery system allows for long-term transgene expression that does not diminish over time.
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The results in the present study suggest that trans-differentiation of BM resident cells into neurons occurs. However, the ultimate proof that such BM cells can migrate to the brain and differentiate into CNS-specific cells may require that the putative cells that undergo trans-differentiation be isolated from the CNS and that functional studies show CNS-specific cell functions.( z' ]" |2 q1 D1 w: F
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Conclusions

These observations suggest that gene delivery to the bone marrow may be a way to provide long-term transgene expression in the neurons, microglia, and astrocytes in the brain, particularly the dentate gyrus. The therapeutic implications of these findings remain to be explored.* S' K* ~* {) Y+ G' f

DISCLOSURES0 k8 o- D) `! ^  O& m* K2 O0 s
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS* M8 n4 P$ a. h' Y6 O
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We appreciate the encouragement and advice of our colleagues Drs. Pierre Cordelier, Kathy Kopnisky, Roger Pomerantz, and Diane Rausch in formulating and executing these studies. This work was supported by NIH Grants MH70287, MH69122, AI48244, and AI41399.
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