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作者：Minoru Tomitaa, Taisuke Morib,c, Kazuichi Maruyamaa, Tasneem Zahira, Matthew Warda, Akihiro Umezawab, Michael J. Younga作者单位：aThe Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA;bDepartment of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo, Japan;cDepartment of Pathology, Keio University, Tokyo, Japan 2 m" p' j5 P+ ?; `; [
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【摘要】' a& d$ U; ^$ ]
Retinal progenitor cells (RPCs) are immature precursors that can differentiate into retinal neurons, including photoreceptors. Recently, it has been reported that bone marrow-derived cells may also be capable of differentiation into cells of central nervous system lineage, including retinal neurons. We compared these two cell types to evaluate their potential as a source of cells for retinal transplantation. Marrow stromal cells (MSCs) and macrophages were isolated from enhanced green fluorescence protein mice. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor to induce neuronal differentiation. RPCs were cultured under the same conditions or with 10% fetal bovine serum. Neuronal marker expression was examined and compared between MSCs and RPCs. MSCs, macrophages, and RPCs were also cultured with explanted retinas from rhodopsin knockout mice to study their potential for retinal integration. MSCs expressed neuronal and retina-specific markers by reverse transcription-polymerase chain reaction and immunocytochemistry. Both types of cells migrated into retinal explants and expressed neurofilament 200, glial fibrillary acidic protein, protein kinase C-, and recoverin. RPCs expressed rhodopsin, a photoreceptor marker we never detected in MSCs. A majority of bone marrow derived-macrophages differentiated into cells that resembled microglia, rather than neural cells, in the explanted retina. This study shows that RPCs are likely to be a preferred cell type for retinal transplantation studies, compared with MSCs. However, MSCs may remain an attractive candidate for autologous transplantation.
1 {9 g, D+ b4 |( G 【关键词】 Bone marrow stromal cells Microglia Retinal stem cells Retinal transplantation Neural differentiation
( B: R5 a/ `8 b4 f! v INTRODUCTION. {( ?6 o2 Z; T' H7 ^, ?
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Marrow stromal cells (MSCs) are a population of multipotent mesenchymal stem cells distinct from hematopoietic stem cells. MSCs were originally reported to contribute to the microenvironment of bone marrow and to be necessary for the proliferation of hematopoietic stem cells .
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9 g- @0 ~- U( j! \The two major clinical subtypes of retinal degeneration (RD) are retinitis pigmentosa and age-related macular degeneration. A hallmark of these diseases is photoreceptor cell degeneration, resulting in visual loss. No effective restorative treatment exists for either RD subtype. Previously, we reported that brain-derived progenitor cells can migrate and differentiate into cells expressing markers of mature neurons and glia when grafted to the retina of mice and rats with RD , we examined the differentiation of macrophages when grafted into the retina. Here, we compared the potential of retinal progenitor cells (RPCs) and MSCs for use in retinal transplantation studies.
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) I. `/ j! ] f0 I) H& ]6 s/ VMATERIALS AND METHODS
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8 u6 ^6 s) W& z' e+ rExperimental Animals8 h& d ^7 E7 a6 [2 r
|4 D( f+ X5 P! q1 Q- y/ Q- ?All experiments were performed in adherence with the ARVO (Association for Research in Vision and Ophthalmology) Statement for the Use of Animals in Ophthalmic and Vision Research and with the Schepens Eye Research Institute Animal Care and Use Committee (Boston, MA). Rhodopsin knockout mice (rho¨C/¨C mice; C57/Bl6 background, provided by Peter Humphries, University of Dublin, Trinity College, Dublin, Ireland) and postnatal day 1 (P1) enhanced green fluorescence protein (EGFP) mice (C57BL/6 background; Dr. Masaru Okabe, University of Osaka, Osaka, Japan) were euthanized by CO2 gas.+ V3 x8 d8 E, J E0 h
, ?; ~) @, F" i. K9 O, s% LIsolation of MSCs and Macrophages! H+ a5 F: h" P# y8 y3 j
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Humeri, femurs, and tibias were obtained from P1 EGFP mice and divided into small pieces. These small pieces were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 with 10% fetal bovine serum (FBS), and the nonadherent cells were removed by replacement of the media. After approximately 2 weeks, the adherent cells became confluent and were incubated with trypsin for 3 minutes and removed from the flask. All cell cultures were maintained at 37¡ãC, 5% CO2.3 Q3 q- T! z E
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After two or three passages, bone marrow-derived adherent cells were incubated with trypsin for 3 minutes to generate a single-cell suspension. Cells (1 x 106) were labeled with phycoerythrin-conjugated antibody against CD11b (1:50, marker for macrophages; BD Biosciences PharMingen, San Diego, http://www.bdbiosciences.com) and Cy-5-conjugated antibody against CD45 (1:50, marker for hematopoietic cells; BD Biosciences PharMingen). To isolate MSCs (CD45¨C, CD11b¨C) and macrophages (CD45 , CD11b ) from bone marrow-derived adherent cells, cell sorting was performed (data not shown). After sorting, the isolated MSCs and macrophages were cultured in 20% FBS for 2¨C3 days and then used for the subsequent experiments.6 Y, O) P, X8 a* {4 l/ `' p0 V
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" ~3 r8 f8 I- Y: u' bRPCs harvested from the retina of P1 EGFP mice were isolated and maintained in culture as previously described .9 i1 A# l& c# N7 X7 ]
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Neural Differentiation and Characterization of MSCs) C3 I5 o: `7 |6 y% J
- M: W, a. C9 x5 F. X, Q* PTo examine the differentiation of GFP-expressing MSCs in vitro, MSCs were incubated with trypsin for 3 minutes to generate a single-cell suspension. Cells (1 x 103) were plated on eight-well poly(D-lysine)/laminin-coated chamber slides (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) in DMEM/F-12 medium supplemented with 25 ng/ml BDNF (R&D Systems, Minneapolis, http://www.rndsystems.com), 40 ng/ml NGF (R&D Systems), and 20 ng/ml bFGF (R&D Systems) and were fixed with 4% paraformaldehyde (PFA) at 2 weeks after plating. The cells were blocked in 1% bovine serum albumin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) 0.2% Triton-100 (Sigma-Aldrich) and then incubated for 2 hours with primary antibody to Ki67 (1:100, cell proliferation marker; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), nestin (1:1, immature neuronal marker; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww/), glial fibrillary acidic protein (GFAP) (1:50, astrocyte marker, Dako), MAP-2 (1:500, neuronal markers; Sigma-Aldrich), anti-protein kinase C (PKC)- (1:200, bipolar cell marker; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), 2D4 rhodopsin (1:500, rod photoreceptor marker; kind gift of Dr. R. Molday, University of British Columbia, Vancouver, BC, Canada), and recoverin antibodies (1:1,000, photoreceptor and bipolar cell marker; Chemicon International, Temecula, CA, http://www.chemicon.com). After rinsing in phosphate-buffered saline (PBS ), samples were incubated in Cy3-conjugated species-specific IgG (1:800) for 1 hour. Samples were rinsed again and then coverslipped in polyvinyl alcohol-1,4-diazabicyclo (2.2.2) octane (PVA-Dabco) with 4',6-diamidino-2-phenylindole (DAPI) and viewed under fluorescent illumination. As a control, the untreated MSCs were fixed with 4% PFA and labeled with the same antibodies.
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. T/ p. k+ j* E {) v5 }Differentiation and Characterization of RPCs# S) w. Y; Z F) |5 G0 J# @
) s B, B4 D/ k4 a9 tTo examine the differentiation of GFP-expressing RPCs in vitro, RPC spheres were incubated with trypsin for 1 minute to generate a single-cell suspension. In two separate experiments, cells (1 x 103) were plated on eight-well poly(D-lysine)/laminin-coated chamber slides (BD Biosciences) in DMEM/F-12 medium supplemented either with 10% FBS or with BDNF, NGF, and bFGF (the same growth factors used in MSCs differentiation experiments ) and were then fixed with 4% PFA at 1 day and 2 weeks after plating. The cells were then reacted and prepared with the antibodies described for MSCs.
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0 @( s& E8 K2 E$ }- \Morphometry of Differentiated Cells
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: D7 U: J+ K( h C) W0 RIn each of the three culture conditions (MSCs with BDNF, NGF, and bFGF; RPCs with 10% FBS; and RPCs with BDNF, NGF, and bFGF), quantitative morphometry was performed by counting positive cells from a total cell number of at least 200 cells per well in randomly selected wells, selected based on DAPI labeling (n = 5). In this counting study, cells (1 x 103) were plated on eight-well poly(D-lysine)/laminin-coated chamber slides (BD Biosciences). Five of eight wells were randomly chosen (by a masked observer), and all cells in the wells were counted. Nestin-positive cells from RPCs were counted at day 1, and MSCs and RPCs positive for other markers were counted after 2 weeks of treatment.$ t: O: n7 C0 E" v. M' b3 `( ?
) ^: o; i* H! ~6 L T6 Q1 ~Reverse Transcription-Polymerase Chain Reaction Analysis of MSCs! r A: ~+ {* V; d
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For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was extracted using TRIzol (Invitrogen) from MSCs grown in the presence or absence of BDNF, NGF, and bFGF in poly(D-lysine)/laminin-coated culture dishes (BD Biosciences) and from P1 EGFP mice retina for a positive control. First-strand cDNA was prepared from total RNA by reverse transcriptase using oligo(dT) primers. To detect nestin, ß-tubulin class III (BT-III; neuronal marker), Map2, GFAP, PKC-, recoverin, and rhodopsin, primers were used as described in Table 1.' i, I9 {- {7 Z! A. D- x
8 a R3 _$ ?5 X$ ^Table 1. Primers used for reverse transcription-polymerase chain reaction analysis& O- o$ [4 j9 a) j1 N
7 Y& N4 v: {+ h/ Q- L$ v$ H# ORetinal Organ Culture
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$ B% J2 u" M2 q6 mRetinal organ culture was performed as previously described with minor modifications. Briefly, eyes were enucleated from rhodopsin knockout (rho¨C/¨C) mice and transferred to ice-cold Hanks' balanced salt solution (Invitrogen). The retinas were separated from the retinal pigment epithelium and placed onto Millicell-CM membrane culture inserts (diameter 30 mm, pore size 0.4 µm; Millipore Corporation, Billerica, MA, http://www.millipore.com) with the ganglion cell layer downward. The inserts with neural retina were placed in six-well plates containing approximately 1 ml/well of medium containing DMEM/F-12 supplemented with B27 neural supplement (Invitrogen), 2 mM L-glutamine (Sigma-Aldrich), 2,000 U of nystatin (Invitrogen), and 100 µl/ml penicillin-streptomycin (Sigma-Aldrich). Organ cultures were maintained at 37¡ãC, 5% CO2 and fed every 2¨C3 days. W8 ~: _% R6 _( I) V& ~
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Explant Coculture5 k( ]# S2 }1 a0 A! F- F; t7 T7 \$ O! Q
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The host retinas were explanted from rho¨C/¨C mice (4¨C8 weeks of age). Cell suspensions (1 µl, 5 x 103 cells/µl) containing (a) RPCs (n = 12); (b) MSCs with (n = 12) or without (n = 6) pretreatment with BDNF, NGF, and bFGF for 1 week; and (c) macrophages (n = 6) were added to the retinas using a pipette immediately after isolation of recipient retinas. We placed the grafted cells onto the surface of retinal explants using a 200-µl pipette. The cells were spread out over the entire surface of the explant, confirmed by viewing under fluorescent illumination. The explanted retinas were cultured for 1 week.
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0 g: ~% q" d8 `" @8 P7 f6 M8 cTissue Preparation) c: x/ \9 j7 ~* I
* U/ l# [$ ^6 f5 H7 ~5 gAfter 1 week in explant coculture, the explanted retinas were fixed with 4% PFA, followed by cryoprotection with 20% sucrose. The retinas were sectioned at 12 µm on a cryostat. Sections were stained with neurofilament (NF) 200 (1:1,000, neuronal marker; Sigma-Aldrich), GFAP, PKC-, recoverin, and rhodopsin antibodies as described above. After fixation with PFA and sucrose, some whole-mount retinas were stained with biotin-Griffonia simplicifolia (GS)-lectin (5 µg/ml, microglia and macrophages marker; Sigma-Aldrich) for 15 minutes and NF200 antibody for 2 hours. After rinsing in PBS, samples were respectively incubated in Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) and Cy3-conjugated species-specific IgG (1:800) for 1 hour. Samples were rinsed again and then coverslipped in PVA-Dabco and viewed under fluorescent illumination.; J# b3 \- T: z8 d" E
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Characterization of MSCs1 \( s" i- N" j1 d* q" R2 U6 B# z
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When grown on conventional substrates in media supplemented with 10% FBS, GFP-transgenic MSCs exhibited high levels of endogenous green fluorescence (Fig. 1A). The untreated MSCs did not express nestin, Map2, GFAP, PKC-, recoverin, or rhodopsin (data not shown). To examine differentiation in vitro, medium without 10% FBS was supplemented with BDNF, NGF, and bFGF. After 2 weeks of culture under differentiation conditions, MSCs differentiated into cells with neuronal morphologies and neurite-like processes (Fig. 1B) and also formed spheres (Fig. 1C). Subpopulations of MSCs expressed nestin (Fig. 1D¨C1F), Map2 (Fig. 1G¨C1I), GFAP (Fig. 1J¨C1L), PKC- (Fig. 1M¨C1O), and recoverin (Fig. 1P¨C1R). These markers are consistent, although not conclusive, with differentiation into retinal neurons. Interestingly, these immunopositive cells also showed morphological evidence suggestive of differentiation into immature photoreceptors, bipolar cell types, glial cells, and neuronal cells (Fig. 1F, 1I, 1L, 1O, 1R). We could not find any rhodopsin-positive cells from treated MSCs.
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Figure 1. Differentiation and characterization of marrow stromal cell (MSCs) in vitro. Undifferentiated GFP MSCs grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, viewed under fluorescein isothiocyanate illumination (A). MSCs cultured in serum-free medium with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor for 14 days (B¨CR). After 2 weeks of culture under differentiation conditions, MSCs morphologically differentiated into neuronal shape and had neuronal processes (B) and also formed spheres (C). Constitutive GFP expression (D, G, J, M, P), antibody/cytokeratin-3 immunoreactivity for nestin (E), Map2 (H), GFAP (K), PKC- (N), and recoverin (Q), and merged images (F, I, L, O, R). Abbreviations: GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; PKC, protein kinase C.
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- @' P& K f3 W) M2 g' B2 I' I6 ? dCharacterization of RPCs
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When grown on conventional substrates in medium supplemented with EGF, GFP-transgenic RPCs exhibited high levels of endogenous green fluorescence (Fig. 2A) and maintained an undifferentiated state characterized by ubiquitous Ki67 and nestin immunoreactivity (Fig. 2B, 2C). Cells could be maintained in this state for up to 1 year or 50 passages as neurospheres. To examine differentiation in vitro, medium without EGF was supplemented with 10% FBS. After 2 weeks culture under differentiation conditions, the cells were analyzed immunocytochemically. The number of Ki67 cells markedly decreased (data not shown), and subpopulations expressed GFAP (Fig. 2D), Map2 (Fig. 2E), PKC- (Fig. 2F), recoverin (Fig. 2G), or rhodopsin (Fig. 2H). These markers are consistent with differentiation into rod photoreceptors, bipolar cells, and Muller glia, all of which are known to be born late in retinogenesis. Moreover, these immunopositive cells also showed morphological evidence suggestive of immature photoreceptor differentiation, as well as of other retinal cell types (Fig. 2D¨C2H).
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Figure 2. Differentiation and characterization of retinal progenitor cell (RPCs) in vitro. RPCs formed green fluorescent protein-positive neurospheres (A). RPCs cultured in the absence of epidermal growth factor and in the presence of 10% fetal bovine serum for 1 (B, C) or 14 (D¨CH) days. The cells were stained for Ki67 (B), nestin (C), GFAP (D), Map2 (E), PKC- (F), recoverin (G), and rhodopsin (H). Abbreviations: GFAP, glial fibrillary acidic protein; MSC, marrow stromal cell; PKC, protein kinase C.
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; {0 K( c7 N% R7 NQuantitative Evaluation of Differentiated Cell Numbers: MSCs Versus RPCs
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To examine the optimal source of cells for retinal transplantation, quantitative evaluation of differentiation into neuronal and retinal cells was carried out using cell counting as previously described .( |# ~% J# b* ~/ G& D; s
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After 2 weeks of BDNF, NGF, and bFGF treatment, the percentages of surviving MSCs expressing nestin, Map2, GFAP, PKC-, and recoverin were 5.55%, 3.27%, 1.42%, 3.97%, and 13.9%, respectively. The percentages of nestin-, Map2-, GFAP-, PKC--, recoverin-, and rhodopsin-positive cells from RPCs treated with 10% FBS were 90.5%, 15.2%, 64.4%, 12.9%, 23.6%, and 3.17%, respectively. The rates of nestin-, Map2-, GFAP-, PKC--, recoverin-, and rhodopsin-positive cells from RPCs treated with BDNF, NGF, and bFGF were 89.2%, 29.4%, 10.9%, 28.2%, 22.3%, and 2.25%, respectively (Fig. 3A).' K& d/ Q& \" d! R
) Y/ }* y: U7 MFigure 3. Comparison of BMSCs and RPCs. (A): The number of cells differentiated into retinal cells: comparison of marrow stromal cell (MSCs) and RPCs. In this study, nestin-positive cells were counted at day 1, and other markers cells were counted at 2 weeks after treatment. (B): Effect of BDNF, NGF, and bFGF on transcription of retinal cell markers. Semiquantitative reverse transcription-polymerase chain reaction analysis was carried out to determine the effect of BDNF, NGF, and bFGF on MSCs. MSCs without treatment showed only weak recoverin expression. (MSCs without treatment did not express nestin, BT-III, Map2, GFAP, PKC-, and rhodopsin completely.) After 2 weeks of BDNF, NGF, and bFGF treatment, treated MSCs expressed nestin, BT-III, Map2, GFAP, PKC-, and recoverin; however, rhodopsin expression was not found. Recoverin expression was increased in treated MSCs. Abbreviations: BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; BMSC, bone marrow stromal cell; bp, base pair; BT-III, ß-tubulin class III; FBS, fetal bovine serum; GF, growth factor; GFAP, glial fibrillary acidic protein; NGF, nerve growth factor; PKC, protein kinase C; RPC, retinal progenitor cell.. o3 D" @9 z, T3 G) d9 f9 h" D
% N: N1 f) Z w& E& u. w' dRT-PCR Analysis of BDNF, NGF, and bFGF Treatment+ Y' P) g5 y7 I2 ^1 U/ p2 w
5 r2 M, i1 c. m8 C3 {Semiquantitative RT-PCR analysis was carried out to determine the effect of BDNF, NGF, and bFGF on MSCs (Fig. 3B). MSCs without treatment showed only weak recoverin expression. (MSCs without treatment did not express nestin, BT-III, Map2, GFAP, PKC-, or rhodopsin.) After 2 weeks of BDNF, NGF, and bFGF treatment, MSCs expressed nestin, BT-III, Map2, GFAP, PKC-, and recoverin. Rhodopsin expression was not found. Recoverin expression was increased in treated MSCs.1 |; n$ I$ |& ]( y v- f6 x
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Macrophages Differentiated into Microglia After Coculture with Explanted Retinas* u3 N F, \1 T+ q2 ~! g
3 E. z8 q0 h5 W- K$ B+ SAfter coculture with explanted rho¨C/¨C mouse retinas, macrophages were viewed by fluorescent illumination at 3 and 7 days. Macrophages migrated into the retina and assumed morphology very reminiscent of microglial cells (Fig. 4A¨C4C). The cocultured macrophages also expressed GS-lectin, a marker of microglia (Fig. 4D¨C4F). There was no evidence of neuronal differentiation upon immunocytochemical and morphological analyses (data not shown).
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Figure 4. Macrophages differentiated into microglia after transplantation to explanted retinas. Rho¨C/¨C mice retina at 3 (A) and 7 (B, C) days. Macrophages migrated into retina and morphologically changed their shape to that resembling microglia (A¨CC). Confocal (D¨CF) images seen at 1 week after grafting; constitutive green fluorescent protein expression (D), macrophage/microglia antibody/cytokeratin-3 immunoreactivity (E), and merged images (F).
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Migration and Differentiation of MSCs2 M4 j: d& B2 Y) l/ h$ `
, e4 A' ]8 J: e- A' EAt 1 week in coculture, MSCs with and without pretreatment of BDNF, NGF, and bFGF migrated into explanted rho¨C/¨C retina (Fig. 5A). MSCs without pretreatment did not show morphological or immunocytochemical evidence of neural differentiation (data not shown). On the other hand, pretreated MSCs showed morphological and immunocytochemical evidence of neuronal differentiation. Pretreated MSCs migrated into explanted retinas (Fig. 5A) and expressed NF200 (Fig. 5B¨C5G), GFAP (Fig. 5H¨C5J), PKC- (Fig. 5K¨C5M), and recoverin (Fig. 5N¨C5P). We also found morphological evidence of neuronal differentiation (Fig. 5B¨C5P). However, we could not find any rhodopsin-positive cells among coculture, pretreated MSCs.- @" P9 ], F! V# n" C
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Figure 5. Migration and differentiation of pretreated marrow stromal cell (MSCs) into explanted retinas of rho¨C/¨C mice. A large number of MSCs migrated into explanted retinas of rho¨C/¨C mice (A). Epi-fluorescent (K¨CP) and confocal (B¨CJ) images of the expression of neural and photoreceptor markers by pretreated MSCs that were grafted onto explanted retinas from rho¨C/¨C mice, seen at 1 week after grafting; constitutive green fluorescent protein expression (B, E, H, K, N), antibody/cytokeratin-3 immunoreactivity for NF200 (C, F) (whole mount), GFAP (I), PKC- (L), recoverin (O), and merged images (D, G, J, M, P). Abbreviations: GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; INL, inner nuclear layer; NF, neurofilament; ONL, outer nuclear layer; PKC, protein kinase C.
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3 H/ {, m+ l5 R- b9 dMigration and Differentiation of RPCs8 L& t# W" w) }. N8 t4 j! @
5 h; m: a+ O! M) |At 1 week in coculture, RPCs migrated into all retinal lamina adjacent to the graft after addition to the outer retina and showed morphological evidence of neuronal differentiation (Fig. 6D¨C6I). GFP donor cells coexpressed a number of markers indicative of phenotypic maturation, including GFAP (Fig. 6A¨C6C), PKC- (Fig. 6D¨C6F), and recoverin (Fig. 6G¨C6I). In the rho¨C/¨C mice, the rod marker rhodopsin was not detected in either grafted RPCs or the host outer nuclear layer.
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# b0 C7 Q" d% x( ~% x$ _& NFigure 6. Migration and differentiation of pretreated retinal progenitor cells (RPCs) into explanted retinas of rho¨C/¨C mice. Confocal images of the expression of neural and photoreceptor markers by RPCs grafting to explanted retinas of rho¨C/¨C mice, seen at 1 week after grafting; constitutive green fluorescent protein expression (A, D, G), antibody/cytokeratin-3 immunoreactivity for GFAP (B), PKC- (E), recoverin (H), and merged images (C, F, I). Abbreviations: GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; INL, inner nuclear layer; MSC, marrow stromal cell; ONL, outer nuclear layer; PKC, protein kinase C.
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2 K! ^4 x! n- _3 {DISCUSSION
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( B7 s$ k5 [- V0 L6 [" V, OThe results presented here demonstrate that MSCs treated with BDNF, NGF, and bFGF can differentiate into retinal cells expressing Map2, BT-III, GFAP, PKC-, and recoverin by immunocytochemistry and RT-PCR. In the explanted retina, pretreated MSCs showed differentiation into retinal cells expressing NF200, GFAP, PKC-, and recoverin, although nonpretreated MSCs did not show any evidence of differentiation into retinal cells. This shows that treatment with growth factors (as in our previous report .! J+ ]7 }' r5 h/ z z5 O
) f6 r) j; ?. AAs with previous studies in the brain , we did not find evidence of differentiation into neuronal or glial cells in our explant study. Further studies will be needed to determine the neuronal potential of macrophages and microglia.' d7 J1 s/ r ]! k @2 ~
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From a clinical perspective, MSCs are a good source for stem cell transplantation. Bone marrow cell transplantation is already an approved therapy for some kinds of hematological diseases and has the advantage of the possibility of autologous cell transplantation. Moreover, because recent reports have shown that MSCs have the capacity to modulate allogeneic cellular immunity , MSCs may be useful for allogeneic transplantation., h) y; u" q. d2 d2 }
& \+ ?# u. Z! j# U5 V( RCell fusion has recently been proposed as the underlying explanation for the apparent plasticity and "transdifferentiation" of stem cells, including MSCs. This raises questions about the mechanisms of transdifferentiation in vitro and in vivo .
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) D& m- ^* n ?CONCLUSION- O5 h, u ~. ]# t/ `& r* a$ y
5 Y. x4 u J. Z4 F7 {" I2 l; J* V' n' |The present study shows that RPCs have clear advantages over MSCs in potential retinal transplantation applications. First, no evidence was found for MSC differentiation into rod photoreceptors. Second, RPCs showed more complete differentiation into retinal cell subtypes than did MSCs, and this occurred at a significantly higher rate. Finally, we have previously reported that neuronal progenitor cells (NPCs) have inherent immune privilege, suggesting increased resistance of allogeneic NPC grafts to host rejection . Such findings suggest the possibility that RPCs possess immune privilege properties as well. MSCs also have significant therapeutic potential in transplantation medicine because they can be readily obtained through a well-established clinical procedure. They are relatively easy to isolate and expand for autologous transplantation without the need for immunosuppression or the risk of rejection. In this comparison study, we submit that RPCs possess significant advantages for differentiation into retinal cells compared with MSCs.
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DISCLOSURES8 B3 A% _0 N/ N8 z3 L1 `2 \
" W/ e4 V+ D3 q1 T% B4 \The authors indicate no potential conflicts of interest., w: u/ x3 U# i0 z
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ACKNOWLEDGMENTS
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: Z3 D/ O0 C0 sThis work was supported by grants from the U.S. Department of Defense, National Institutes of Health (09595; M.J.Y.), and by a gift from Richard and Gail Siegal. We thank Prof. Susumu Ikehara (Department of Pathology, Kansai Medical University, Osaka, Japan) for advice.8 p' f) w2 y$ F# I( A S
【参考文献】
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发表于 2015-6-18 19:50
