作者：Anand S. Srivastavaa, Steve Shenoudaa, Rangnath Mishrab, Ewa Carriera作者单位：aMoores University of California San Diego Cancer Center, Department of Medicine, University of California San Diego, San Diego, California, USA;bDivision of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA 8 f5 h( a0 q* J. g& j
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+ }8 F" T) g4 T' E! Z4 F+ T 【摘要】% `7 X8 N# A* q
An understanding of feasibility of implanting embryonic stem cells (ESCs), their behavior of migration in response to lesions induced in brain tissues, and the mechanism of their in vivo differentiation into neighboring neural cells is essential for developing and refining ESC transplantation strategies for repairing damages in the nervous system, as well as for understanding the molecular mechanism underlying neurogenesis. We hypothesized that damaged neural tissues offer a niche to which injected ESCs can migrate and differentiate into the neural cells. We inflicted damage in the murine (C57BL/6) brain by injecting phosphate-buffered saline into the left frontal and right caudal regions and confirmed neural damage by histochemistry. Enhanced yellow fluorescent protein-expressing ESCs were injected into the nondamaged left caudal portion of the brain. Using immunohistochemistry and fluorescent microscopy, we observed migration of ESCs from the injection site (left caudal) to the damaged site (right caudal and left frontal). Survival of the injected ESCs was confirmed by the real-time polymerase chain reaction analysis of stemness genes such as Oct4, Sox2, and FGF4. The portions of the damaged neural tissues containing ESCs demonstrated a fourfold increase in expression of these genes after 1 week of injection in comparison with the noninjected ESC murine brain, suggesting proliferation. An increased level of platelet-derived growth factor receptor demonstrated that ESCs responded to damaged neural tissues, migrated to the damaged site of the brain, and proliferated. These results demonstrate that undifferentiated ESCs migrate to the damaged regions of brain tissue, engraft, and proliferate. Thus, damaged brain tissue provides a niche that attracts ESCs to migrate and proliferate. 2 W0 R$ E" k) O5 M4 t
【关键词】 Transplantation Mouse embryonic stem cell Brain Embryonic stem cell 7 [. Q& ]! U6 P2 T# f8 A4 P' j7 N INTRODUCTION 2 ^' I) {$ a* S( l * a4 Z$ D+ {. r+ P' XEmbryonic stem cells (ESCs) are nontransformed, pluripotent cells that are derived directly from the inner cell mass of preimplantation embryos .& |+ i2 Z% \, [' i) p
5 w+ B( Y7 C5 _6 e4 D6 `9 S# ]0 C6 GHere, we demonstrate in vivo proliferation and migration of ESCs toward the damaged portions of the brain. Proliferation of the ESCs was confirmed by histochemistry and by monitoring the expression of genes related to proliferation of ESCs by using real-time polymerase chain reaction (PCR). We have demonstrated an increased number of transplanted ESCs in the brains of the mice by immunohistochemistry and their migration toward the sites of injury artificially induced in other sites of the brain. We have also demonstrated that the levels of stemness genes like Sox2, Oct4, and FGF4 first increase with the increasing number of cells and then decline as the implanted ESCs possibly differentiate into the neighboring types of cells. Further studies are needed to understand how the damaged brain tissue-specific niche induces differentiation of ESCs.2 }8 M1 W% T" |- a
8 ~ C. d9 z# V2 mMATERIALS AND METHODS& n$ z1 c( y! G3 u8 H4 T( n
% c! _/ u5 d3 }' N \Induction of Neural Degeneration # {4 h, r2 m0 _. a' a 2 v) g, e* S# T4 [% zC57BL/J6 mice purchased from The Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) were maintained and used in accordance with University of California San Diego Animal Care and Use Committee guidelines. Mice were anesthetized by isoflurane inhalation and placed in a stereotaxic frame. A burr-hole mark was made at the place of injection according to Ikeda et al. . Eighteen animals were used to generate neural degeneration using PBS. The nature and degree of neural damage inflicted by PBS injection were regularly confirmed by immunohistochemistry., {! G1 {) T% |/ n$ F! U* T0 k
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ESC Culture $ a- z8 V J& K' C6 g! ]. O- D/ V( b1 B a, c( B
The ESC-enhanced yellow fluorescent protein (EYFP) cells were obtained from transgenic mice expressing EYFP reporter gene .7 ]4 e, r9 N6 `& B2 j8 [" x
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ESC Transplantation! j: w) \) @# }9 `! X
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Animals were anesthetized with isoflurane inhalation. Each animal received a 1.0-µl injection of either vehicle (PBS) alone or suspension of 5,000 ESCs into one site of the right striatum (from the bregma: anterior 1.0 mm, lateral 3.0 mm, ventral 5.0 and 4.5 mm, incisor bar 0) using a 10-µl Hamilton syringe (Reno, NV, http://www.hamiltoncompany.com) fitted with 22-gauge needle. Coordinates were set according to the atlas of Bjorklund et al. . A 2-minute waiting period was allowed for ESCs to settle before the needle was removed. Each animal received 5,000 ESCs in 1 µl of vehicle. Six animals were used in each experimental group. ; ?% h' k* r" J! t' O# Q4 |, Q 7 C! h! P2 C% r& cHistological Procedures 7 n7 z2 f6 I5 m6 x5 p 8 E: o; S7 m0 H; ?% tFor histological studies, animals were anesthetized terminally by an intraperitoneal overdose of pentobarbital (150 mg/kg). Animals were perfused intracardially with 100 ml of heparin saline (0.1% heparin in 0.9% saline) followed by 200 ml of 4% paraformaldehyde solution in PBS. Brain tissues were post-fixed for an additional 8 hours in the same fixative and then equilibrated with 20% sucrose in PBS. Forty-micron-thick sections were cut on a freezing microtome, and tissue sections were collected in PBS . r2 y9 ?% Y$ p C + m* z. b1 S! wImmunohistochemistry of ESC-Injected Brain# B) _' C+ P7 g' y1 w
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Brain sections were subjected to the light and fluorescence microscopy for the detection of EYFP ESCs in the mouse brain. To avoid a false signal generated by autofluorescence, the sections were subjected to the secondary YFP peroxidase staining using anti-YFP antibody. Migration of the ESCs was studied by staining the brain section for platelet-derived growth factor receptor (PDGFR) using mouse anti-PDGFR-specific antibody (BD Biosciences PharMingen, La Jolla, CA, http://www.bdbiosciences.com/pharmingen) . In brief, the brain sections were rinsed in PBS and then preincubated in 4% normal bovine serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) for 60 minutes. For YFP staining, specimens were reacted with monoclonal anti-YFP antibody, and for PDGFR staining sections were stained by anti-mouse PDGFR antibody followed by a secondary horseradish peroxidase-conjugated polyclonal antibody (BD Biosciences PharMingen).2 O5 x/ ^( u! h. f0 s8 @
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Real-Time PCR u+ {, ?, T6 s) q. C# u$ D
6 P" B) x9 N C7 g( j) S, FTotal RNA was isolated using the Qiagen RNeasy kit (Germantown, MD, http://www1.qiagen.com) according to the manufacturer¡¯s protocol. Regular PCR reactions were carried out with Taq DNA polymerase (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), and level of the transcripts was assessed by agarose gel electrophoresis. Real-time PCR was performed with AmpliTaq Gold polymerase in a PerkinElmer Biosystems 5700 thermocycler (PerkinElmer Life and Analytical Sciences, Boston, http://las.perkinelmer.com) using the SYBR Green detection protocol as outlined by the manufacturer with two-step thermal profiles (i.e., denaturation at 95¡ãC for 15 seconds followed by elongation at 60¡ãC for 1 minute as described by Mishra et al. . Statistical significance between different groups of mice was calculated by Student¡¯s t test.8 y N- k @; B l6 o- d
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7 S4 |% z/ \$ u6 i5 M6 E8 j$ }3 o# y2 CExperimental Brain Injury by Excessive PBS Injection, s/ r2 }5 q5 O, ?6 v y
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Several reports are available regarding creating experimental brain injury using liquid nitrogen and mechanical damage of brain cells . We inflicted the brain damages using excessive PBS injection as described in Materials and Methods. First, we monitored progression of damage inflicted by injection of 2.5 µl of PBS in the brain tissues of the mice. Twenty-four and 72 hours after the injection, brain was subjected to morphological and histochemical examination to study the degeneration of neurons. We noticed an apparent degeneration of brain cells only after 72 hours of PBS injection; this was evident by the presence of a black patch of dead cells in the portion of the brain injected with PBS.% o- A0 \5 }/ e( q
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Implanted ESCs Survive in the Brain * T( v- y- T, X- a0 @0 G* U , X2 C! K, ?7 `After the implantation of ESCs, we monitored their survival in the new surroundings. Twenty-four hours after the implantation, survival of ESCs was confirmed by immunohistochemical staining for YFP. As seen in Figure 1A, the presence of cells positive for YFP indicated that implanted cells survived in the damaged regions of the brain. As expected, the newly implanted cells were few and confined to the site of implantation. Findings of the fluorescence studies carried out on the brain sections confirmed that the YFP-positive staining was from the newly implanted cells only (Fig. 1B). 6 \8 ]4 P9 W4 V5 ?' Q 8 G. W- y0 n; W" MFigure 1. Yellow fluorescent protein (YFP) embryonic stem cells (ESCs) after 24 hours of injection into the murine brain. (A): Sections of mice brains were stained for YFP using horseradish peroxidase (HRP)-labeled secondary antibody. Dark brown color shows the presence of YFP-expressing ESCs (arrow). (B): Confocal fluorescence microscopic image of sections of mouse brains. Magnification x400 (arrow). * [# L" i$ f+ M1 v( _# @; b% T; r4 F; q. q
Migration of ESCs Toward the Damaged Region of Brain 1 Q8 {( @% M( C4 S* V$ P# @ M* T ) \4 e' p$ e. a& E/ y8 k/ @One week after injection of ESCs, their survival, proliferation, and migration was determined by immunostaining. Fluorescence and immunohistochemical analysis of tissue sections, including ESCs implantation sites, was carried out. A higher number of cells positive for YFP was recorded, showing that implanted cells not only survived but also proliferated in the new biological paradigm (Fig. 2). Results obtained by immunostaining for YFP were confirmed by the findings of fluorescence analysis also (Fig. 2). Histological studies of the PBS injected sites of the brain were carried out to determine whether the implanted ESCs migrated toward the injured cell sites. We detected the ESCs at the damaged portion of brain after 1 week of injection (Fig. 3), indicating the fact that newly implanted cells migrated toward the damaged tissues. Furthermore, we used PDGFR staining as an additional marker to determine whether the ESCs survived in the brain. Brain transplanted with ESCs expresses a higher level of PDGFR. The immunohistological analysis of PDGFR expression also confirmed the ESC survival and migration (Fig. 4) . 6 I! A L% `/ M! O8 f0 k( ] * X$ c7 N& Z5 OFigure 2. Histological sections showing embryonic stem cell (ESC)- and PBS-injected sites in the murine brain after 1 week of injection. (A): Superimposed image of light and fluorescent microscope. (B): Fluorescent microscope photomicrograph. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline. 4 N" }! C0 N# C0 B+ Y% r1 L, R - j. s' l7 p# @7 }Figure 3. Yellow fluorescent protein (YFP) embryonic stem cells (ESCs) migrate toward the damaged portion of the brain. Photomicrograph shows the peroxidase staining for YFP-expressing ESCs (brown color). Magnification x40. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline. v/ C' q9 h) g! U* S" f7 U$ B3 }+ W * `# | }* q8 o6 L8 v( y* OFigure 4. Immunohistochemistry staining with anti-PDGFR (purple) antibody was performed on 10-µm sections of a murine brain injected with embryonic stem cells (ESCs) and damaged with phosphate buffered saline (PBS). (A): PDGFR expression after ESC and PBS cell injection in the left and right portions of the brain, respectively. The PBS injection site has initiated a dynamic environment where cell migration mechanisms are highly expressed relative to the ESC injection site. (B): The edges of the ESC injection site containing cells that express high levels of PDGFR. Abbreviations: ES, embryonic stem; PDGF, platelet-derived growth factor. 2 O) u9 q; X. b1 ^1 M ]7 A' C8 c; V. C# h& ~0 q( w- W$ o/ R
Expression of Stemness Genes (Sox2, Oct4, and FGF4) in the ESC-Injected Brain5 y- m8 Q, f9 e: Z9 P# [
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Expression of a group of genes is needed to maintain the stem cell-like character of pluripotent stem cells, and this group of genes is known as stemness genes. Sox2, Oct4, and FGF4 are known as stemness genes . The real-time PCR analyses of these genes showed approximately fourfold upregulation in the expression of Sox2, Oct4, and FGF4 genes in comparison with that in the non-ESC-injected portion of the brain (Fig. 5). Furthermore, the expression of these genes increased to their maximum level after the 1 week of ESC injection at the site of the dead portion of the brain (Fig. 6). $ B9 H' I P- Z" d' X) l- s* d& t, ?# f2 e
Figure 5. Real-time polymerase chain reaction analysis of genes (Oct4, FGF4, and Sox2) involved in neural cell development in noninjected, embryonic stem cell (ESC)-injected, and PBS-injected site of the murine (C57/Bl6) brain. After 3 days of injections, samples were collected and gene expression studies were performed as described in Materials and Methods. The ESC-injected site shows an almost fourfold increase in the FGF4 expression and twofold increase in Oct4 and Sox2 expression in comparison with PBS 2.5 (damaged portion), PBS 1.0 (PBS control), and control (noninjected portion) of the brain (n = 6). *p value ' | j6 i. F k) J! h3 [( b$ [ 8 P9 `' z- {& K) b. ~Figure 6. Real-time polymerase chain reaction analysis of genes (Oct4, FGF4, and Sox2) involved in neural cell development in phosphate-buffered saline (damaged brain)-injected site of the murine (C57/Bl6) brain. After 24 hours, 1 week, and 1 month of injections, samples were collected and gene expression studies were performed as described in Materials and Methods. The embryonic stem cell-injected portion of brain showed an almost fivefold increase in FGF4 expression and fourfold increase in Oct4 and Sox2 expression in comparison with those at 24 hours. Expression of these genes returned to a normal level after 1 month (n = 6). *p value ' r. D9 s8 w$ [4 \& }% J
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DISCUSSION . ?7 ]) W: l; w0 M7 K {' `! W$ g1 o1 E4 m! M1 y3 E
We hypothesized that degenerating neural tissues secrete some chemical signals that stimulate ESCs to migrate toward the damaged portion of brain and also that the ESCs in new biological environment differentiate into the neural stem cells. To test this hypothesis, we microinjected YFP-expressing ESCs away from the damaged sites in the murine brain and monitored their survival and migration profile.9 Z: _- R( P1 t# Y: X0 V
3 ^/ E/ r6 d3 e' T) E) zImmunohistochemistry results demonstrated that 24 hours after the injection, the ESCs were still localized at the site of microinjection, and we were able to detect a very faint signal of YFP from the injected cells. Moreover, we did not detect any ESCs at the damaged portion of the brain. However, after 7 days of ESCs implantation, we detected a far stronger signal of YFP in comparison with that of day 1. The fluorescence signals indicate an accumulation of ESCs at the periphery of the damaged portion of the brain. It was evident by the overexpression of PDGFR protein from the same place. These results emphatically demonstrate that the ESCs settle and survive in the new biological setup after the injection into the mouse brain and, more importantly, migrate toward the damaged portion of the brain.+ h4 t+ f: u# m7 q, `
7 s* z& ]! F8 T4 _# X. MIt is well documented that Oct4, Sox2, and FGF4 work in a coordinated manner to maintain the pluripotent nature of ESCs also have correlated the upregulation of these three genes with the proliferation of ESCs and downregulation of these genes with the differentiation of neural stem cells. Therefore, we speculate that the downregulation of these three genes after 1 month of ESC injection may be due to differentiation of ESCs into the neural cells, possibly of neighboring types. Further extensive analyses are necessary to elucidate the molecular mechanism surrounding the up- and downregulation of these genes after transplantation into the murine brain.5 [2 D4 D; g$ \# t; J9 `. i
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CONCLUSION e" h- s) w/ L$ H" f9 B+ p( o) N: m1 E) i: g( _- Q: `5 E# ]" ?
The results of our histological and gene expression studies suggest that ESCs proliferate and migrate to the damaged portion of the murine brain after transplantation and possibly differentiate into the neural stem cell. ) O# Y* N1 N& y+ r 9 m$ ?! `/ Q7 \DISCLOSURES 8 ]# C2 k9 ?' H( c* W: d; ^5 k3 ^! _* L! p: W, [4 ?2 I% `
The authors indicate no potential conflicts of interest. ( ?3 A- M9 T- R, j 【参考文献】2 E1 r& J0 \% d& ]
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Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372¨C376.作者: mcflyqiang 时间: 2013-4-8 17:58