Ionizing Radiation Enhances the Engraftment of Transplanted In Vitro–Derived Mul - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, Florida, USA( g, }# W5 x2 _

Key Words. Green fluorescent protein ? Irradiation ? Multipotent astrocytic stem cell ? Neurosphere ? Neuroblast ? Neural stem cell ? Subependymal zone: L1 D! t8 `' c" c! {
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Correspondence: Gregory P. Marshall II, Ph.D., 1600 SW Archer Road, Gainesville, Florida 32611, USA. Telephone: 352-392-0231; Fax: 352-392-0025; e-mail: gpm2@ufl.edu8 L; D# E0 H+ u8 K
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ABSTRACT4 m8 }" S; `  i5 A

Adult neurogenesis is limited to two well-characterized regions of the mammalian brain: the subgranular layer of the hippocampal dentate gyrus and the forebrain subependymal zone (SEZ) . The former produces neurons that functionally integrate into the granular cell layer of the hippocampus, whereas the SEZ produces neuroblasts in the walls of the lateral ventricles (LVs) that migrate along a defined pathway, known as the rostral migratory stream (RMS), to the olfactory bulb (OB), where most die but some differentiate and functionally integrate into the existing cytoarchitecture as granule or periglomerular interneurons . These migrating neuroblasts have been well characterized and are known to be immunopositive for both the pan-neuronal marker ?-III tubulin and the active neuronal migration marker polysialylated neuronal cell adhesion molecule (PSA-NCAM) . The number of newly generated neurons produced daily in the SEZ of the adult mouse has been estimated at 30,000, leading many to conclude that a self-renewing stem cell must reside in the mouse SEZ for this rate to be sustained for the life of the animal . The cell type in the SEZ believed to be the neural stem cell (NSC) has been identified as a slowly dividing astrocyte known as the type-B cell , and NSCs can be isolated from the adult SEZ and cultured in vitro to form spherical clones known as neurospheres (NSs) , which are capable of producing the major cell types of the neural lineage (neurons, astrocytes, and oligodendrocytes) upon differentiation.8 W5 j. X2 a8 Q3 j/ s6 x
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NSs are capable of increasing in number after repeated passages in culture while retaining both their multipotency (self-renewal) and the ability to integrate into host neural tissue upon transplantation , leading many to believe that the NS-forming cell is the in vitro correlate of the in vivo NSC. Multipotent astrocytic stem cells (MASCs) isolated from the SEZ of neonatal mice have also been identified as an in vitro correlate of the NSC, displaying the potential not only to generate multipotent NSs but also to integrate into host neural tissue upon transplantation in much the same way as the NS .
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Transplantation of NSs or MASCs derived from animals transgenic for the reporter gene encoding green fluorescent protein (gfp ) into the LV of normal adult C57BL/6 mice results in minimal engraftment, with few donor-derived neuroblasts present in the RMS or OB. Transplantation into neonatal mice, however, results in relatively robust levels of engraftment with comparatively high numbers of donor-derived neuroblasts and interneurons present in the OB weeks after transplantation. This presents a quandary in the research of adult NSCs because neonatal and adult brains are dramatically different with respect to graft receptivity. A method for enhancing the engraftment of donor cells into adult neurogenic regions is crucial to a better understanding of the differentiation and integration potential of grafted cell types.: N$ W" @0 c5 w( t4 P" i, U0 J
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Perhaps the most well-characterized adult stem cell is the hematopoietic stem cell (HSC), which, in addition to the aforementioned stem cell characteristics of pluripotency and self-renewal, is also capable of long-term reconstitution of the entire hematopoietic system of a myeloablated animal. In fact, ablation of endogenous HSCs by exposure to high doses of ionizing radiation is critical for facilitating maximum engraftment of transplanted HSCs, which cannot normally compete with the native HSCs for access to the stem cell niche .* \+ ?$ x7 y) F: b, O6 N0 V$ K
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Depletion of the NSC niche has been previously achieved with antimitotic agents  and both ionizing and x-irradiation , yielding transient and long-term depletion of neurogenesis in the hippocampus and SEZ. Neurogenesis in the hippocampus can be attenuated by exposure to varying levels of x-irradiation, as seen by a decrease in the number of migrating granular neurons out of the subgranular layer . Previous studies investigating the effects of focused exposure of x-irradiation to the brain of adult rats on SEZ neurogenesis have reported ablation of NSCs in the SEZ immediately after irradiation . A dose-dependent recovery of NSCs occurred within 2 months of exposure, with recovery never reaching basal levels in all but the lowest doses. Little has been done to characterize the effects of a single, whole-body lethal or sublethal dose of ionizing radiation on SEZ neurogenesis and subsequent engraftment of transplanted in vitro–derived NSCs in the adult mouse.4 c4 |* y! f# @7 e* C

We show here that a single, lethal dose of ionizing radiation significantly depletes the SEZ, RMS, and OB of migrating neuroblasts and that this depletion persists at 3 months after irradiation. The SEZ of lethally irradiated (LI) mice contains fewer neurosphere-forming cells than untreated, age-matched mice, indicating that the stem cell pool of the SEZ has been adversely affected by the radiation. LI renders the SEZ unreceptive to gfp  MASCs transplanted into the LV, as no donor-derived cells were observed in the OB after transplantation, in contrast to engraftment seen in control mice.; I3 ^# |% A8 L' a6 k
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Exposure to milder levels of radiation resulted in a transient decrease in mitotic SEZ neuroblasts, with donor-derived MASC engraftment into the OB at significantly higher levels than seen in controls. These results offer a potential model system for the analysis of NSC candidates in the adult animal, allowing researchers to more easily determine the engraftment potential of candidate NSCs." t" K! u/ H8 \3 i' Z' I1 X: Q
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MATERIALS AND METHODS

Effects of LI on Migrating Neuroblasts in the RMS

The migration of neuroblasts through the RMS from the SEZ to the OB is visible as a robust chain of PSA-NCAM–positive cells (Fig. 2A). Two weeks after LI, mice show a marked decrease in the number of PSA-NCAM–positive neuroblasts in the RMS (Fig. 2B). This observation was corroborated after staining of tissue sections from the brains of both wild-type (WT) and LI mice with the pan-neuronal marker ?-III tubulin (data not shown). Neuroblast depletion is variable, with some animals retaining small pockets of cells in the RMS. However, the overall abundance of migrating neuroblasts in the RMS of LI mice is never similar to those seen in untreated mice.1 `+ S% W% V: L3 W9 C8 g! Q) D
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Figure 2. Effects of lethal irradiation on migrating neuroblasts in the RMS of adult C57BL/6 mice. Mice were subjected to 850 rad of x-irradiation and then supplied with a rescue dose of wild-type bone marrow to allow for recovery of the ablated hematopoietic system. At 2 weeks after irradiation, the animals were perfused with 4% paraformaldehyde and their brains sectioned into 40-μm-thick sagittal sections. Antibodies against the migrating neuroblast-specific marker PSA-NCAM were applied to the tissue, and the sections were photographed at x10 magnification. (A): In nonirradiated control animals, PSA-NCAM–positive neuroblasts are abundant and can be seen extending from the ventricle to the olfactory bulb. Inset in (A) depicts the RMS and migrating neuroblasts at x40 magnification. (B): The RMS of lethally irradiated animals is noticeably devoid of migratory neuroblasts. Inset in (B) displays the depleted RMS (arrows) at x40 magnification. Abbreviations: OB, olfactory bulb; PSA-NCAM, polysialylated neuronal cell adhesion molecule; RMS, rostral migratory stream; V, lateral ventricle.
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Furthermore, the volume of migrating neuroblasts does not recover to the level seen in control animals, even at 3 months after LI (data not shown). These findings led to the conclusion that exposure to lethal levels of ionizing radiation results in permanent damage to the neuroblast-producing cell of the SEZ.1 `, Q0 D" q6 ~$ ]; w
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Analysis and Quantification of Neuroblast Depletion in the SEZ by LI
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To quantify the degree of neuroblast depletion induced by LI, we used BrdU to label mitotic cells within the SEZ . The SEZ in WT animals immunolabeled with antibodies against ?-III tubulin and BrdU on serial coronal sections, at the level of the anterior commisure, contains a robust layer of dividing neuroblasts (Fig. 3A). Three weeks after LI, this same region exhibits a significant decrease in newly generated neuroblasts (Fig. 3B), and this depletion persists at 3 months after LI (Fig. 3C). A decrease of approximately 60% in the number of BrdU-positive cells was observed at the 3-week time point compared with the control. Student’s t-test analysis confirms that this decrease is significant (p = .003). There is an 87% decrease in the number of BrdU-positive cells at the 3-month time point compared with control (p = .002). The 68% decrease in the number of BrdU-positive cells between 3 weeks and 3 months after LI was also significant (p = .03) (Fig. 3D)." \/ X. ?/ j/ p) @/ t

Figure 3. Incorporation of BrdU by neuroblasts in the SEZ of control and LI adult C57BL/6 mice. (A–C): photographic montages generated from x10 magnified images of sections stained for BrdU (red). (A): Nonirradiated control animal. (B): Age-matched littermate 3 weeks after LI. (C): Littermate 3 months after LI. Insets display double-labeling of neuroblasts with both BrdU (red) and ?-III tubulin (green) antibodies at x40 magnification. (D): Total number of BrdU-positive neuroblasts in three adjacent coronal sections of either control or LI mice was tabulated and placed into the above graphical format for quantification of the number of BrdU-positive SEZ neuroblasts calculated as percent of control. Three weeks after LI (n = 4), there are approximately 60% fewer BrdU-positive neuroblasts than in nonirradiated controls (n = 4, p = .003). Three months after LI, this decrease is slightly greater, at 87% (n = 4, p = .002). *Significant values in difference between LI and control animals. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; LI, lethal irradiation; SEZ, subependymal zone.- n( `/ ~; I+ n; e
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?-III Tubulin immunolabeling confirms that the cells scored are, in fact, neuroblasts rather than other mitotic cells residing in this area (insets in Figs. 3A–3C). These data suggest that the decrease in migrating neuroblasts in the RMS is reflected in the SEZ and that this depletion is significant and long-term.5 \. d" f& O. P2 E% [

Effects of LI on NS Yield in Culture

As it is generally accepted that the NS-forming cell is the in vitro manifestation of the NSC , we cultured NSs from both control and LI mice in a blind-study format to determine if the stem cell pool in the SEZ was affected by the LI. NSs cultured from LI brains displayed an average decrease in yield of approximately 77% compared with the wild-type cultures (Fig. 4; p = .02). This decrease closely corresponds to the decreased levels of BrdU-positive neuroblasts in vivo after lethal irradiation, further supporting the validity of this finding. It has been reported that the stem cell population of the SEZ is between approximately 0.02% and 1.0% of the total cells , as determined by NSs yield from dissociated SEZ tissue, and the average yield of NSs from the control brains in this study falls within this range (0.15%, data not shown). The average yield of NSs isolated from the LI brains was significantly lower, at 0.03%, indicating that exposure to lethal doses of radiation depletes the number of NSCs in the brain responsible for the generation of NSs.; ]" K! A% n" `) o  H# {, i: a7 \
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Figure 4. Effect of LI on neurosphere cultures isolated from adult SEZ. SEZ tissue was cultured to generate neurospheres from both nonirradiated control adult mice and LI adult mice (2 months survival, n = 3), with all tissues treated identically. The resulting neurosphere yield was determined according to the protocol described in Materials and Methods. The LI culture yielded an average of 77% fewer neurospheres than observed in control cultures (significant decrease; p = .02; indicated by *). Abbreviations: LI, lethal irradiation; SEZ, subependymal zone.' D2 u$ w" f- R8 D) f8 j" n  N1 D
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LI Attenuates Engraftment of Transplanted gfp  MASCs7 C1 O( d3 F$ V* m' V9 |

Three weeks after irradiation, LI animals were transplanted with gfp  MASCs into the LV and the cells were allowed to engraft for 3 weeks. Whereas nonirradiated controls contain a small but consistent number of donor-derived migratory neuroblasts in the OB, no migratory cells are present in the OB of LI mice (n = 5).  [2 w7 v7 g0 R0 B) B$ i. A2 x- L8 Y

It is possible that the lethal dose of ionizing radiation inhibited functional engraftment of the transplanted cells (possibly by inducing irreparable damage to the radiosensitive support cells of the SEZ), so a lower exposure dose of 450 rad was assayed as a milder form of injury for the enhancement of gfp  MASC engraftment.8 S& E+ L3 m0 `0 S% f* ^9 m& ~7 U
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Analysis of RMS Neuroblast Migration and Quantification of SEZ Neurogenesis After SLI3 k# L7 b! }  G% t
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Unlike LI, SLI (450 rad) does not completely abolish hematopoiesis in the bone marrow of adult mice, and SLI animals do not require a rescue dose of bone marrow for survival. Low levels of focused gamma irradiation (1–3 Gy) have been shown to result in a transient increase in mitotic cell activity in the SEZ, with levels eventually diminishing in the weeks following in a dose-dependent fashion compared with untreated controls . Contrary to the results after LI, we observed no decrease in the overall number of ?-III tubulin–positive migratory neuroblasts in the RMS of irradiated animals 3 weeks after whole-body exposure to 450 rad (a dose equivalent to 4.5 Gy). BrdU quantification assays revealed that although mitotic cell activity in the SEZ was significantly decreased in the hours immediately after irradiation, the levels returned to near normal at 3 weeks and eventually increased to above control levels at 6 weeks (Fig. 5). Blind analysis of NS yield from SLI brains at 3 weeks after SLI revealed a 30% decrease in the number of NSs compared with nonirradiated controls (Fig. 6; p = .032), with control NSs yield again falling into the aforementioned reported range of 0.2%–1.0% (0.12%, data not shown).6 {% |1 E% H, T$ W6 ], \  t2 l2 t

Figure 5. Incorporation of BrdU by neuroblasts in the SEZ of control and SLI adult C57BL/6 mice. (A–C): photographic montages generated from x10 magnified images of sections stained for BrdU (red). (A): Nonirradiated control animal. (B): Age-matched littermate 6 hours after SLI. (C): Littermate 3 weeks after SLI. Insets display double-labeling of neuroblasts with both BrdU (red) and ?-III tubulin (green) antibodies at x40 magnification. (D): The total number of BrdU-positive neuroblasts in three adjacent coronal sections of either control or SLI mice was tabulated and placed into the above graphical format for quantification of the number of BrdU-positive SEZ neuroblasts calculated as percent of control. Six hours after SLI, there are approximately 83% fewer BrdU-positive neuroblasts than in nonir-radiated controls (p = .001, n = 3). This depletion persists 24 (78%, p = .001, n = 3) and 48 (90%, p = .001, n = 3) hours after SLI. Three weeks after irradiation, the number of mitotic SEZ neuroblasts recovers to near control levels, and it eventually increases to 13% above control levels at 6 weeks (p = .003, n = 3). *Significant values in difference between SLI and control animals. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; SEZ, subependymal zone; SLI, sublethal irradiation.
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Figure 6. Effect of SLI on neurosphere cultures isolated from adult SEZ. SEZ tissue was cultured to generate neurospheres from both nonirradiated control adult mice and adult mice SLI 3 weeks prior (n = 4), with all tissues treated identically. The resulting neurosphere yield was determined according to the protocol described in Materials and Methods. The cultures derived from SLI brains yielded an average of 30% fewer neurospheres than did identically treated cultures derived from nonirradiated control mice (significant decrease; p = .032; indicated by *). Abbreviations: SEZ, subependymal zone; SLI, sublethal irradiation.
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SLI Significantly Enhances Engraftment of Transplanted gfp  MASCs
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Three weeks after irradiation, SLI animals were transplanted with gfp  MASCs into the LV and the cells were allowed to engraft for 3 weeks. In a portion of the irradiated animals (Fig. 7D–F), a fourfold increase in the number of gfp  migratory cells in the OB was seen, as compared with nonirradiated controls (n = 14) (Fig. 7A–C). Taken as a whole, the average number of gfp  migratory cells in the OB of SLI animals was twice the average number seen in nonirradiated controls (significant at p = .014) (Fig. 7G), indicating that SLI significantly enhances the engraftment potential of transplanted gfp  MASCs, albeit in a variable fashion.
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Figure 7. SLI significantly enhances the engraftment potential of gfp  MASCs. Three weeks after irradiation, passage-three gfp  MASCs were transplanted into the lateral ventricles of control and SLI animals at the following coordinates: A-P, –0.2; M-L, –1.2; H-D, –2.5. Sagittal brain sections were collected 2 weeks after transplantation and stained with ?-III tubulin (red). (A, D): x20 photographic montages depicting the OB of a nonirradiated control animal (A) and SLI animal (D) (gfp filter). Insets are x40 images of engrafted neuroblasts residing in the OB proper (red, ?-III tubulin; green, gfp). x63 confocal imaging reveals donor-derived migratory neuroblasts (green) present in the OB surrounded by ?-III tubulin–positive (red) neurons of both (B, C) control animals and (E, F) SLI animals. Scale bar in (B) and (E) = 40 μm. Quantification of 14 transplanted animals per condition revealed the presence of approximately twice as many donor-derived migratory neuroblasts in the OB proper of SLI animals compared with nonirradiated controls (G) (p = .014, significance indicated by *). Abbreviations: MASC, multipotent astrocytic stem cell; OB, olfactory bulb; SLI, sublethal irradiation.

DISCUSSION

This research was supported by NIH grants NS37556 (to D.A.S.), HL70143 (to D.A.S.), and NS041472 (to E.D.L.).! Q$ y7 g' V2 z$ x5 F3 h7 z) @
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DISCLOSURES
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The authors indicate no potential conflicts of interest.8 z( K  D7 y/ u6 ?% O
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