Neurogenic Stem Cells Have the Capacity to Disperse Widely and Fuse with Host Neurons in Adult Rats

Research Article

J Stem Cell Res Transplant. 2014;1(2): 1012.

Neurogenic Stem Cells Have the Capacity to Disperse Widely and Fuse with Host Neurons in Adult Rats

Emily Kimes, Michele Kanemori, Daniella Amri, Ashley Noone, Jerika Barron, Ashley Saito, Omar Cortez-Toledo, Ellie Cortez-Toledo, David Arrizon, Johanna Quist, Yohualli Balderas, Melissa Miranda, Tina Tran, Frances Kim, and Kerry Thompson*

Occidental College, VA Greater Los Angeles Healthcare Center, University of California, Los Angeles, USA

*Corresponding author: Kerry Thompson Ph.D, Occidental College, VA Greater Los Angeles Healthcare Center, University of California, Los Angeles, USA

Received: September 01, 2014; Accepted: October 17, 2014; Published: October 20, 2014


Neural stem cells; Fusion; Transplantation; Cortical pyramidal cells; Purkinje cells


The central nervous system (CNS) has a relatively limited capacity for neurogenesis, and self-repair. As a result, diseases that produce neural losses are particularly devastating. The greater the amount of neural tissue loss, the greater the impact on the functioning of the patient. Traumatic brain injury and degenerative CNS diseases such as Alzheimer's disease, Parkinson's disease, and intractable temporal lobe epilepsy (TLE), are clinical challenges that could benefit from the development of cell replacement therapies. For these patient populations, the ability to replace lost brain tissue with functional neurons and glia, is an ambitious, but worthwhile goal. Embryonic stem cells (ESC) that can be genetically engineered, propagated and differentiated, or "neuralized", for subsequent transplantation into the central nervous system, would offer a means to accomplish this important goal [1-5].

Rapid progress is being made in the field of ESC-derived neuron generation and characterization [6]. In most studies, ESC are exposed to a series of chemical treatments that push their differentiation down a neural pathway that can be characterized biochemically. Many related studies have demonstrated proof-of-principal for the transplantation of these cell lines into animal model systems [7-9]. It has been reported that neuralized ESC that have been genetically engineered with reporter molecules, remain viable in the host for long periods [10] and, remarkably, that fully differentiated neural phenotypes expressing transgenes can be found in the host within 30d of transplantation [11]. These findings are important milestones toward the development of neuron replacement therapy, but the interpretation of these studies is challenged by recent reports of the capacity for ESC-derived neurons to fuse with host neurons.

The concept of stem cell fusion in the brain is not new to the literature. In fact, early reports of what was initially believed to be bone marrow derived stem cell (BMDC) trans differentiation in the cerebellum [12] was later discovered to be either complete [13-15] or partial [16] fusion of BMDC with fusionogenic cerebellar Purkinje cells. In the absence of tissue damage BMDC fusion occurs only rarely, and it is not found in brain cells outside of the cerebellum. Presumably because these are rare events, and seemingly specific to BMDC and Purkinje cells, the phenomena has not been consistently pursued as a potential contributor to the histological outcomes following ESCderived neural cell transplantation. Recently, however, studies have shown that fusion does, in fact, occur following transplantation of ESC-derived neural cells and, similar to the BMDC studies, particular cell types seem to be more fusionogenic [11-17].

We have been pursuing transplantation strategies for the treatment of intractable neurological diseases like TLE using genetically modified neural cells [18-22]. In our previous studies using neural cell lines of non-stem cell origin, we have never seen evidence of widespread dispersion or fusion [20-23]. We report here that genetically modified neurogenic stem cells have the capacity to disperse widely following cerebral transplantation into the striatum, and to fuse with host neurons. As has been recently reported [11-13], cortical pyramidal cells are highly fusionogenic, but we believe the fusion-competent cells are not limited to only this cell type. These data are consistent with transplantation results extending back many years and may call for a re-interpretation of those data to include potential fusion events.

Materials and Methods

Cells and cell culture

The feeder free ZHTc6 cell line (24) was purchased from the Institute for Stem Cell Research (Edinburgh, UK). This line was created by genetically engineering wild-type CGR8 murine embryonic stem cells to constituitively express a transactivator molecule (tTA), and one allele of the Pou5f1 gene (which codes for Oct-3/4) under the transcriptional control of a tTA-sensitive promoter (hCMV*- 1). The Oct-3/4 expressing construct is bicistronic and also codes for the reporter molecule beta-galactosidase (β-gal) fused to a neomycin resistance gene driven by the same promoter. In this cell line, up-regulation of OCT-3/4 expression (in the absence of tetracycline), combined with the removal of serum, and leukemia inhibitory factor (LIF), has neurogenic effects [25]. The cells were cultured on gelatinized plates in media containing the following: Glasgow Minimum Essential Medium (Sigma), 2 mM glutamine, 1 mM sodium pyruvate (Gibco), nonessential amino acids (Gibco), 10-15% (v/v) fetal bovine solution (Invitrogen), 1:1000 dilution of β-mercaptoethanol (Sigma), and 500-1000 units per ml of LIF, (Millipore) and grown on 0.1% gelatinized plates and then incubated at 37.0 C and 7.0% CO2 until islands formed. Media was replaced daily. In vitro differentiation was performed by eliminating LIF and growing the cells in media with 4% serum (modeling transplantation into the host).

In vitro histochemistry and immunocytochemistry

To stain stem cell cultures, cells were grown to 70-90% confluence in 6-well dishes and then washed and fixed in a 0.25% glutaraldehyde solution for 15-30 min. The fixative was removed; the cells were washed thoroughly and then incubated at 37.0oC in 1ml of an X-gal solution prepared according to the manufacturer's recommendations (Promega Corporation). The solution was removed 1-18 hrs later, and then the cells were washed and analyzed using an inverted scope and regular light microscopy. In some differentiation experiments, cells were fixed, permeabilized, and incubated with an anti-Nestin antibody (Chemicon International) followed by exposure to a fluorescein-conjugated secondary antibody.


Sprague Dawley females were purchased from Taconic Biosciences Inc. (Hudson, NY). Animals were 200-250 grams (approximately 2-3 mo (N=35)). Rats were housed under a 12-h light/ dark cycle with free access to food and water. All procedures were performed in accordance with the IACUC guidelines approved by Occidental College.

Unilateral 6-hydroxydopamine (6-OHDA) lesion

In a subset of animals, dopaminergic neurons were unilaterally targeted by focal administration of 6-OHDA into the medial forebrain bundle (MFB). Rats were anesthetized with a mixture of xylazine: ketamine (10mg/kg:85mg/kg) or with isoflurane vapor using a vaporizer. Rats were injected with 25 mg/kg (i.p.) desipramine, 30 minutes before 6-OHDA injection, in order to prevent uptake of 6-OHDA by noradrenergic neurons. Stereotaxic surgery was performed to deliver 6-OHDA unilaterally at two locations within the MFB: 2.5μl (0.5μl/2min) AP -4.4, ML -1.2, DV -7.8, and 2.0 μl (0.5 μl/2min) AP -4.0, ML -0.8, DV -8.0, relative to bregma [26-28]. 6-OHDA was administered as 3 μg/μl in a 1% ascorbic acid solution. To minimize oxidation, 6-OHDA solutions were freshly made, kept on ice, and light-protected. Animals were kept on a 37.0?C warming pad during surgery and in a 37.00C recovery chamber after the surgery. A solution of 5% dextrose was injected subcutneously after surgery at 10% body weight.

Five to six days after surgery rats were behaviorally screened for the extent of the 6-OHDA lesion using the amphetamine rotation test. Rats were placed in a Rotomax rotometer (AccuScan Instruments, Inc., Columbus OH), which consists of a 12" diameter Plexiglas cylinder on a flat surface. A harness extends down and permits the animals to touch the wall of the cylinder. Animals were injected with 5 mg/kg amphetamine sulfate and the number turns/5 minutes were calculated from net ipsilateral turns over 30 minutes following a five minute habituation period. Peak responses were anticipated within this time period [29]. Rats that rotated at least 5 ipsilateral (to lesion) turns per minute (net) were designated as "positive responders" and animals that rotated below that level were designated "negative responders". The negative responders were used as an additional "non-lesioned"control group. Animals were returned to their home cages for a period of 2-4 weeks before they received stem cell transplantation into the striatum that was targeted for denervation.


On the day of surgery, animals were injected with a combination of xylazine and ketamine or exposed to isoflurane vapor to produce surgical anesthesia. Once the animals were placed into the stereotaxic apparatus they were injected with neurogenic ESCs unilaterally into the striatum. The majority of the animals were injected with undifferentiated cells to promote cell survival after transplantation. Three of animals were transplanted with cells that were grown under differentiating conditions for three days. The cells were washed and suspended in sterile PBS at 150K cells/μl and held on ice for no longer than two hours. Three injections were made into the striatum at the following coordinates relative to bregma: AP 0.0, ML -1.2, and DV -5.5, 4.5 3.5 with 1.0 μl delivered over a two minute period to each site. Prior to injection of ESC, the surgery needle was washed with ethanol followed by thorough PBS washes. Animals were kept on a 37.00C warming pad during surgery and then placed in a 37.00C recovery chamber after the surgery until they regained their righting reflex. The surgical controls that were used for comparison were injections of dead cells (exposure to hypertonic conditions, N=4), predifferentiated cells (LIF removal and serum reduction, N=3) and current and historical PBS controls (N=1 and N=4 respectively


At multiple pre-determined survival times after the transplantation of cells, the animals were transcardially perfused with cold 4% paraformaldehyde or a 2% paraformaldehyde and .5% glutaraldehyde mixture (for EM processing) and the brains (and liver samples were removed and cryoprotected in 30% sucrose. Brains were recovered 24hr (N=3), 3 days (N=3), 7days (N=3), 2 wks (N=3), and 4wks (N=18 (13) lesioned and (5) unlesioned). The tissue was serially sectioned using a cryostat by blocking and cutting the whole cerebrum (coronal plane), cerebellum (sagittal plane), and liver samples by taking slices at 20, 40, or 60 depending on the histological protocol. Adjacent sections were used for hematoxylin and eosin (H&E) staining and X-gal histochemistry (each 20 μ) and were typically taken every 200μ with adjacent 40μ or 60μ sections taken for immunohistochemistry or TEM, respectively. Tissue was sampled more frequently in the plane of the cell injection to maximize observations in the region of highest interest, which was the transplanted striatum and the surrounding cortex. For H&E staining and X-gal histochemistry, sections were cut onto glass slides. For immunohistochemistry and TEM, sections were placed into PBS.

X-gal Histochemistry

β-gal positivity was used to analyze and locate the β-gal-positive cells in the brain and liver tissue sections. Glass-mounted sections of both brain and liver, and 60μ free-floating sections of the brain, were pretreated by permeabilization with 0.01% deoxycholic acid and 0.02% Igepal in PBS at RT. The slides were then thoroughly washed and the mounted sections were encircled using a PAP pen or grease, so that each of the sections could be incubated in a filtered X-gal solution containing 5 mM potassium ferrocyanide, 5mM potassium ferricyanide, 2mM MgCl2, and 250 μg/ml X-gal in PBS (pH 7.4 or 8.5 to discriminate bacterial β-gal from endogenous enzyme sources) within a dark box placed within a humidified chamber and 37oC. The sections were left to incubate overnight. Sections were then rinsed, mounted onto glass slides if necessary and cover slipped the next morning. Tissue sections were left to dry and then evaluated with conventional light microscopy. Negative controls were incubated for equivalent periods.


X-gal stained tissue was used to quantify areas and numbers of β-gal-positive cells. The tissue section with the largest representation of the injection track, from each of the lesioned-transplanted animals, was used to assess the cross-sectional area of the transplants in the injected striatum. The sections were viewed using a 4X objective and digital images were taken. The ImageJ program was used to outline the perimeter of the transplant and then to calculate the area (N=3 for 1, 3, 7, and 14d, and N=5 for 1 month). Additionally, in the one month post transplant animals, the number of β-gal-positive Purkinje cells was counted in lobes III, IV, and V of the cerebellum [30]. Comparable 1mm sections were targeted in the selected lobes and the total number of β-gal-positive cells were compared to the total number of Purkinje cells in those regions.


Indirect immunofluorescence and immunoperoxidase histochemical strategies were performed on free-floating sections. A polyclonal anti β -gal antibody (Chemicon International) was used at [1:1000], an anti-GFAP antibody (Millipore) was used at [1:1200] and an anti-NeuN (Millipore) antibody was used at [1:500]. Speciesappropriate secondary antibodies were used with either fluorescein or Texas red fluorescence (Vector Labs). For double labeling experiments, antibodies were exposed serially to the tissue sections. In some cases, following the secondary antibody exposure, the sections were exposed to Hoechst (1:1000) to fluorescently label all nuclei. For immunoperoxidase staining, an ABC kit (Vector Labs) was used in accordance with the manufacturer's instructions. In all cases, tissue sections were incubated in the primary antibodies overnight at 4°C.

Immunohistochemical controls included the omission of the primary antibodies, the secondary antibodies, and, in some cases, the inclusion of an endogenous biotin-blocking procedure (Vector Labs). All of these sections were examined by conventional light and fluorescent microscopy and a subset of the tissues were evaluated using a confocal microscope (Leica TCS-SP2 AOBS inverted confocal Microscope (UCLA core facility).

Transmission electron microscopy

A modification of a previously published procedure combining X-gal histochemistry and TEM [16] was used. For a subset of animals at the 30 day survival time point, frozen sections were cut at 60 and then the free-floating sections were reacted with the X-gal solution overnight. The reacted tissue was mounted onto glass slides and then viewed under a dissecting microscope. Cortical sections near the plane of injection, with identified X-gal-positive cells, were excised with a razor blade and then processed for TEM (5-7 excisions per animal). The excised tissue was rinsed for 10 min in 0.1M Na cacodylate buffer and then in 1% osmium made in the same buffer, for 2 hrs. After a second short rinse, the tissue was dehydrated in graded alcohols and then infiltrated with Spurs overnight. The next day the tissue was placed into molds and then baked for at least 24 hrs. Thick sections (1.5) were cut from blocks using a LKB Nova ultramicrotome, they were then counterstained with toludine, and viewed under a light microscope. Blocks producing thick sections that contained cells with evidence of X-gal precipitate and/or with profiles that were consistent with heterokaryon, were selected for further analysis by obtaining thin sections (70nm) that were evaluated using a Zeiss EM 109 transmission electron microscope.


We confirmed that the ZHTc6 line expresses Oct 4 and SSEA-1 (a marker associated with neural stem cell precursors), in the absence of doxycycline (data not shown). In proliferation media, the cells did retain undifferentiated phenotypes, but a fraction would flatten, and many of those cells extended processes. In differentiation media the majority of the cells underwent changes in morphology with stereotypic neuritic projections. Cell division halted after ~3 days in differentiation media and a significant percentage of cells died over a period of five days. The neuritic projections of the adherent differentiated cells, at five days, were positive for the CNS progenitor marker nestin (Figure 1E). The ZHTc6 cells and all of their derivatives, express β-galactosidase in the absence of doxycycline (Figure 1B). This made visualization of the transplanted cells possible by: 1) exposing histological sections to the substrate X-gal which produces a blue reaction product only within the enzyme-containing cells and 2) performing immunohistochemistry using anti-β-galactosidase antibodies. We used both of these techniques simultaneously in all animals. We found that the overall patterns of cellular staining were the same, but that immunohistochemistry was more sensitive as it revealed more cells and their processes (Figures 3 and 4). We also performed nissl staining on adjacent sections for every animal.