Survival and Long Distance Migration of Brain-Derived Precursor Cells Transplant - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:22 |只看该作者 |倒序浏览 |打印

a Wallenberg Retina Center, Department of Ophthalmology, Lund University Hospital;
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b Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Lund, Sweden% K) H5 L! e1 n+ n

Key Words. Brain-derived precursor cells ? RN33B ? Subretinal transplantation ? Cell markers ? Whole-mounted retina ? Migration+ F# ?" Y- ~* _; B& F7 ]  |, h' W

Anita Blixt Wojciechowski, M.D., Department of Ophthalmology, Lund University Hospital, S-221 84 Lund, Sweden. Telephone: 46-46-2220769; Fax: 46-46-2220774; e-mail: a.blixt@bredband.net or anita.blixt_wojciechowski@oft.lu.se) }5 p+ s9 G/ b0 R" }" J5 j

ABSTRACT
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The two major groups of retinal degenerations in humans are known as retinitis pigmentosa and age-related macular degeneration. The diseases within these two groups are very heterogeneous, with varying onsets, severity, and other general clinical manifestations, but they all have two things in common: A) they manifest degeneration of photoreceptor cells, and B) there is no effective treatment available.$ ^1 J' l: b" q

During the second half of the 1980s, the field of experimental retinal tissue transplantation expanded greatly . The method aims at reconstructing the degenerating retina by subretinal grafting of healthy pieces of retinal tissue, which, upon integration with the host retina, will form a functional unit. A few clinical trials using such retinal transplants have been performed, but so far only limited improvement of visual function has been noted .

Instead of using retinal tissue transplants, in vitro expanded multipotent neural precursor cells could serve as donor cells. Currently, protocols are well established for genetic and epigenetic expansion of rodent and human embryonic and adult neural precursor cells. In several experimental intracerebral transplantation studies, such cells have shown a great capacity to survive, migrate, and differentiate into region-specific neurons with appropriate axonal projections as well as to form astrocytes and oligodendrocytes .* m7 ~* ?' ]  O5 A

Retinal transplantation of precursor cells has been performed in order to investigate the possibilities to replace diseased retinal cells and/or to rescue diseased retinal cells in situ by cell-mediated delivery of survival factors. Indeed, transplantation of various neural precursor cells into the retina has been carried out with good cell survival in the normal neonatal and adult host retina , degenerating retina , and injured retina .

To some extent, migration of grafted precursor cells, such as adult rat hippocampal progenitor cells and neonatal rat retinal precursor cells, into the host retina has been observed . We recently described extensive migration in a long-term transplantation experiment using the cell line RN33B, revealing in retinal sections the distribution of RN33B cells from one eccentricity of the adult host retina to the other . Although good retinal graft survival has been estimated by several investigators, no exact number of transplanted cells has been presented, probably due to technical limitations. However, attempts have been made to quantify the survival of grafted cells on cryoprotected sections of the host . In the present study, the brain-derived precursor cell line RN33B was further studied. The cells are genetically immortalized with the temperature-sensitive simian virus (SV40) large T antigen. In order to detect the cells post-grafting, the RN33B cell line is genetically labeled with the reporter genes LacZ and the green fluorescent protein (GFP) .

The purpose of the present study was primarily to estimate survival and assess migration, but also to study integration and differentiation of grafted RN33B cells in whole-mounted retinas.
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MATERIALS AND METHODS' Y. |0 N% t$ j1 _

Similar results in the 3-week and 8-week animals on survival, migration, integration, and differentiation were obtained independent of the different transplantation sessions (Table 1). Therefore, the 3-week and the 8-week groups with the same survival time were pooled.
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Survival of the Transplanted RN33B Cells

Post-transplantation ?-Gal- and GFP-expressing cells were found in 68% (19/28) and 35% (11/31) of the grafted animals at 3 and 8 weeks, respectively (Table 1). A large fraction of the transplanted cells coexpressed ?-Gal and GFP (Fig. 2A and 2B). Images superimposed on each other showed ?-Gal/GFP double-labeling of transplanted cells, as well as subpopulations expressing either ?-Gal or GFP (Fig. 2C and 2D). Smears of the RN33B cells taken prior to transplantation revealed that close to 100% of the cells expressed endogenous GFP (Fig. 1). In control animals, untreated or injected with HBSS medium alone, neither ?-Gal nor GFP immunoreactivity were found.

Figure 2. RN33B cells 3 weeks post-transplantation demonstrating immunoreactivity to (A) ?-Gal (red) and (B) GFP (green). Both A and B are from the same retinal area. The precursor cells have differentiated with variable morphologies and size. Most of the grafted cells have developed long, slender cell somas and processes. The processes have branched and beaded processes. A few cells only express GFP (arrows). C) A retina with double labeling 3 weeks post-transplantation. Cells expressing either ?-Gal (small arrow) or GFP (large arrow), as well as cells expressing both markers (arrowhead), are demonstrated. D) A retina with double labeling 8 weeks post-grafting. Cells expressing ?-Gal (small arrow) or GFP (large arrow), as well as cells expressing both markers (arrowhead), are observed. E) RN33B cells immunoreactive to anti-SV40 (red) 8 weeks post-transplantation. The poor quality of the retina is caused by the pretreatment in 2M HCl before anti-SV40 was used. Both cell markers of RN33B, GFP, and ?-Gal are labeled green. More SV40-expressing cells than marker-expressing cells are revealed, indicating a downregulation of the marker genes. Scale bars = 30 μm./ F# w! U! H1 V7 O, C- x% g

With the stereological method of analysis, cell counting of six retinas with the most intensive endogenous GFP fluorescence and the widest distribution area of grafted cells from the 3-week group revealed an average of 2,838 (± 2,625) ?-Gal-immunoreactive cells and of 1,351 (± 1,045) GFP-positive cells (Table 2). At 8 weeks, the ?-Gal expression was too weak for an appropriate analysis, and quantification of ?-Gal-expressing cells was only possible in one retina out of six, which displayed 31,985 ?-Gal-expressing cells. The average number of GFP-expressing cells in retinas with the most intense fluorescence was estimated as 22,359 (± 10,058) at 8 weeks (n = 6), which indicates that from 3 weeks to 8 weeks post-grafting, the average number of transplanted GFP-expressing cells had increased about 16 times.

Four of the grafted retinas (3 weeks, n = 2 and 8 weeks, n = 2) were exposed to anti-?-Gal and FITC-conjugated secondary antibodies in order to reveal the total population of grafted cells expressing one or both transgenes. The retinas were then further immunoprocessed using anti-SV40 and Texas Red secondary antibody and analyzed for coexpression of ?-Gal/GFP and SV40. Cells were observed immunoreactive to ?-Gal/GFP and SV40, as well as cells only immunoreactive to SV40 (Fig. 2E). Six of the 20 retinas with no detectable ?-Gal or GFP immunoreactivity from the 8-week group were exposed to anti-SV40; no SV40 immunoreactivity was found. Control retinas (n = 2) injected with the HBSS medium alone and untreated retinas did not express SV40 antigen.
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Distribution and Migration of the Transplanted RN33B Cells

With the semiautomatized cell counting system, the distribution area of GFP-positive grafted cells could be delineated. The population of GFP-positive cells was easier to distinguish, thus the distribution area of grafted cells was delineated according to these cells. Since the distribution area of the ?-Gal-expressing cells overlapped the area of GFP-expressing cells at both survival times, this was not considered to introduce a significant bias. The total adult retinal area was estimated to an average of 60.17 (± 7.84) mm2 using values obtained from the nine retinas measured (Table 2). At 3 weeks, RN33B cells were found on a restricted retinal area of, at most, 12.57 mm2 (mean, 5.55 ± 3.75) and on an area of up to 40.8 mm2 (mean, 28.96 ± 11.85) 8 weeks following grafting (Table 2). This correlates to a migratory capacity of the RN33B cells to be distributed throughout a maximum of 21% and 68% of the entire host retina at 3 and 8 weeks, respectively.! ]0 x! z2 _2 G/ x) ]5 p
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Although the RN33B cells migrated over long distances, a large fraction of the grafted cells was found at or close to the implantation site at both 3 and 8 weeks post-grafting. Aggregates of marker-expressing cells were found in the surrounding retina and single scattered cells were seen far from the implantation site (Fig. 3).1 d" A# k* u3 B2 o
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Figure 3. Whole-mounted retina 8 weeks post-transplantation with GFP-expressing RN33B cells (white). A-J represent parts of the schematic drawing of a whole-mounted retina (the boxes in the drawing are larger than the representing digital pictures). Close to the implantation site (black arrow) many cells are demonstrated (C, D, E, and G), and far from the implantation site single scattered cells are revealed (A and H, white arrow). Scale bar = 120 μm.) b9 U5 Y* |! b9 h: x. g
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A distinctive migration pattern was found in four of the grafted retinas at 8 weeks, with RN33B cells migrating as chains forming tubule-like structures (Fig. 4). These cells had morphologies of migrating cells, with elongated cell bodies and an apical process in the direction of the migration . A leading process was demonstrated on both single migrating cells as well as on the cell tubules. The chains of transplanted cells were often arranged radial to the optic nerve head but not along the great retinal vessels (Fig. 5).
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Figure 4. Chains of migrating RN33B cells at 8 weeks after grafting. These long, slender GFP-expressing cells seemed to climb along each other. A leading process of the whole tubule can be seen. Scale bar = 30 μm.
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Figure 5. Higher magnification of Figure 3D. Near the optic nerve head (ON), single cells and chains of grafted cells are arranged radially (arrows) and oriented towards the optic nerve. Scale bar = 120 μm.+ F) H& H% P  o$ p$ ^
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Differentiation and Integration of the Transplanted RN33B Cells# n/ \9 y6 v6 a3 T
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Previously, both the GFP and ?-Gal reporter genes have been reported to be highly useful for morphological analysis of grafted neural precursors . However, in this cell line, GFP allows for a more fine-detailed detection of structural elements such as dendritic spines, terminal boutons, and axonal projections than ?-Gal . In agreement, also in the present study, GFP was found to be more informative concerning cell morphologies than ?-Gal (Fig. 2A).+ C* }7 L; ?& r7 L" L: Z

Both at 3 weeks and 8 weeks post-transplantation, the RN33B cells displayed highly variable morphologies and sizes (Figs. 2C and 2D, 6A and 6B). Differentiated RN33B cells did not differ in morphology between the 3- and 8-week retinas (Fig. 2C and 2D). The grafted cells adapted both neuronal and glial-like morphologies. The cellular profiles included bipolar-like cells with small round or long slender somas and multipolar cells with different sizes of the cell somas. Most of the cells had varying numbers of processes, many of them being branched with multiple neuronal-like spines (Figs. 2B, 6C, and 6D). The cell processes were often in close contact with each other, indicating a cell-to-cell contact (Figs. 2C, 2D, and 6C).4 ?. W! P% ~; s1 t$ v$ O) J0 c

Figure 6. RN33B cells 3 weeks post-transplantation on two different retinal areas. A) ?-Gal-expressing cells (red); B) GFP-expressing cells (green). Different morphologies can be detected, from rounded to long, slender cell somas. Most of the cells have developed long branched processes. Scale bars = 60 μm. C) RN33B cells 3 weeks post-transplantation. The branched processes of the grafted cells are often in close contact with each other (arrow). The processes are beaded or have small spines. Scale bar = 30 μm. D) RN33B cells 3 weeks post-grafting demonstrating ?-Gal (red) and one double-labeled cell (yellow) with four branched and beaded processes. Scale bar = 20 μm. E) RN33B cells expressing ?-Gal (red) 8 weeks post-transplantation on a whole-mounted retina that has been sectioned. Most of the cells are integrated into the OPL and IPL. Scale bar = 60 μm.

In addition, the grafted cells were observed in different layers of the retina, which was revealed using varying focus levels in the microscope. From previous studies , it is known that grafted RN33B cells migrate and integrate mainly into the outer plexiform layer (OPL) and the inner plexiform layer (IPL) of the retina. The same distribution pattern was observed when one whole-mounted retina was sectioned (Fig. 6E).

DISCUSSION- g% i. y! V! P  d
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The present study demonstrates that the precursor cell line RN33B transplanted to adult intact retina survives at 3 weeks in 68% and at 8 weeks in 35% of the animals. However, more cells were found on each retina after 8 weeks compared with 3 weeks. The actual number of grafted cells is most likely not demonstrated because of downregulation of the two marker genes soon after grafting. The amount of SV40 large T antigen-expressing cells in the host retina gives an impression of a far greater survival of cells than revealed by the ?-Gal and GFP expression. It is also demonstrated that the grafted cells display a widespread migration from the implantation site at longer survival times and that the grafted cells differentiated to neuronal- and glial-like phenotypes.5 v: ?3 i- H! Z' j4 `

Still little is known about the factors that influence precursor cell survival, proliferation, and differentiation upon retinal and brain transplantation, therefore multipotent precursor cell transplantation to the retina is a valuable tool in investigating these issues. The possibility to introduce genes ex vivo to modify the precursor cells to produce different factors necessary for the diseased host retina gives an additional perspective. This emerging science will clearly be useful to the obstacles involved in retinal reconstruction.( l( [% u& z9 C8 \5 E+ l; E

ACKNOWLEDGMENT
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* Dr. Englund is now with H. Lundbeck A/S, Department of Neurodegenerative Disorders, Valby, Denmark.
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