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Keywords:

  • Transplantation;
  • Mouse embryonic stem cell;
  • Brain;
  • Embryonic stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

Embryonic stem cells (ESCs) are nontransformed, pluripotent cells that are derived directly from the inner cell mass of preimplantation embryos [1]. These cells have the inherent capacity to participate fully in embryonic development when reintroduced into the blastocyst. In vitro, ESCs can give rise to cell types of all three primary germ layers in a way that recapitulates events of embryogenesis [2]. Therefore, ESCs provide a unique cellular system for experimental dissection of lineage specification and determination. Moreover, understanding and controlling ESC differentiation will be an important step toward harnessing their potential to differentiate in any cell type of need for biomedical purposes. It has been many years since evidence was first obtained for the presence of multipotent precursor cells in the vertebrate and invertebrate nervous systems [3, 4]. Identification of the multipotent cells was evident by the developmental plasticity of central nervous system (CNS) stem cells after the transplantation into ectopic sites in the CNS [5, [6], [7]8]. The potential of CNS stem cells expanded in vitro was also defined [9, [10], [11]12]. The molecular mechanisms controlling CNS stem cell numbers and fate choice are now the focus of work in many laboratories using both in vivo and in vitro systems [13, [14], [15], [16], [17], [18]19]. During development, precursor cells in the nervous system assume a new positional identity within a spatial coordinate system that has a dorsal-ventral, rostral-caudal, and left-right axis [20]. These cells subsequently undergo mitotic arrest, rapidly acquiring characteristics of a specific terminal fate. The differentiation of stem cells in vitro shows that many of these specific features occur in the absence of the precise organization that occurs in vivo. The self-organization shown during stem cell differentiation raises the intriguing question of how these concerted responses are achieved. Expression of several genes is needed to maintain the pluripotent nature of stem cells. The group of genes responsible for maintaining the pluripotency of cells is known as stemness genes, and Sox2, Oct4, and FGF4 are prominent members of the group [21, [22], [23], [24]25]. Several lines of evidence indicate that Sox genes are key players in the determination of cell fates in diverse developmental processes. Sox2 is expressed within the developing neuroepithelium of vertebrate embryos and has been implicated in the maintenance of neuroepithelial cell character. It has also been reported that Sox2 with Oct4 and FGF4 works in a coordinated manner to maintain the pluripotency of ESCs [21, 23].

Here, 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

Induction of Neural Degeneration

C57BL/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. [26]. We induced damage in the murine (C57BL/6) brain by injecting 2.5 μl of phosphate-buffered saline (PBS) into the left frontal and right caudal regions. Coordinates were set according to the atlas of Bjorklund et al. [27]. 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.

ESC Culture

The ESC-enhanced yellow fluorescent protein (EYFP) cells were obtained from transgenic mice expressing EYFP reporter gene [28, 29]. Unless otherwise stated, all reagents for ESC culture were purchased from StemCell Technologies (Seattle, http://www.stemcell.com). In brief, ESC-EYFP cells were maintained in gelatinized dishes in Dulbecco's modified essential medium supplemented with 15% selected fetal calf serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 150 μl, 1 M β-mercaptomethanol, and 10 ng/ml leukemia inhibitory factor according to Hadjantonakis and Nagy [30].

ESC Transplantation

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. [27] and Hoehn et al. [31]. 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.

Histological Procedures

For 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 [32].

Immunohistochemistry of ESC-Injected Brain

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) [33, 34]. 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).

Real-Time PCR

Total 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°C for 15 seconds followed by elongation at 60°C for 1 minute as described by Mishra et al. [35]). Sequences of specific primers for murine genes were as follows: Sox2 (sense: 5′-GGC AGC TAC AGC ATG ATG CAG GAG C-3′, antisense: 5′-CTG GTC ATG GAG TTG TAC TGC AGG-3′), Oct4 (sense: 5′-GAC AAC AAT GAG AAC CTT CAG GAG-3′ antisense: 5′-CTG AGT AGA GTG TGG TGA AGT GG-3′), FGF4 (sense: 5′-CCG GTG CAG CGA GGC GTG GT3′, antisense: 5′-TAC TTC CAG TGG GTG AAG GAA GG 3′), and β-actin (sense: 5′-TTC GTT GCC GGT CCA CA-3′, antisense: 5′-ACC AGC GCA GCG ATA TCG-3′) [36]. Level of transcript was determined by using the 2(−δδC[T]) method as described by Mishra et al. [35]. Statistical significance between different groups of mice was calculated by Student's t test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

Experimental Brain Injury by Excessive PBS Injection

Several reports are available regarding creating experimental brain injury using liquid nitrogen and mechanical damage of brain cells [26]. 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.

Implanted ESCs Survive in the Brain

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).

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Figure Figure 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 ×400 (arrow).

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Migration of ESCs Toward the Damaged Region of Brain

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) [34].

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Figure Figure 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.

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Figure 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 ×40. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline.

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Figure Figure 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.

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Expression of Stemness Genes (Sox2, Oct4, and FGF4) in the ESC-Injected Brain

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 [21, [22], [23], [24]25]. 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).

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Figure 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 < .05. Abbreviations: ES, embryonic stem; PBS, phosphate-buffered saline.

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Figure 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 < .05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

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.

Immunohistochemistry 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.

It is well documented that Oct4, Sox2, and FGF4 work in a coordinated manner to maintain the pluripotent nature of ESCs [21, 23, 24]. Downregulation of these genes has been shown to trigger the differentiation of neural stem cells [24, 36–38, [37], [38]]. Therefore, we selected these important stemness genes (Oct4, Sox2, and FGF4) and studied the expression pattern of these genes in the temporal and spatial manner after the transplantation of the ESCs into damaged murine brain. We observed a significantly higher expression of all three stemness genes (Oct4, Sox2, and FGF4) in the RNA isolated from the ESC injection site of the brain in comparison with the controls (PBS injected portion and noninjected normal brain). Significantly higher expression of these genes suggested that the ESCs proliferated and remained undifferentiated for a significant duration after the transplantation into the murine brain. Our results are consistent with the previous reports of Okuda et al. [23] and Zhao et al. [21], who suggested that the stable expression of Oct4, Sox2, and FGF4 is essential for ESCs to maintain their undifferentiated and differentiated potentials [21, 23]. We also observed an increased expression of FGF4, Sox2, and Oct4 after 1 week of ESC injection into the RNA sample collected from damaged sections of the brain in comparison with the 24-hour sample. The expression level of these genes returned to a normal level after the 1 month of the injection of the cells. Several previous reports suggest that the sustained and higher (more than 50% increase over the normal differentiated ESCs) level of gene expression of Oct4, Sox2, and FGF is essential for maintaining the pluripotent nature of ESCs. However, even if essential, upregulation of these genes would be necessary only at a critical period. Downregulation of these genes after a critical period is important for normal differentiation of neural stem cells from ESCs [21, 23]. Avilion et al. [36] 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.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

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.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References

The authors indicate no potential conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. References