Mesenchymal Stem Cells from Human Extra Ocular Muscle Harbor Neuroectodermal Differentiation Potential

Mesenchymal stem cells (MSC) have been proposed as suitable candidates for cell therapy for neurological disorderssince they exhibit good neuronal differentiation capacity. However, for better therapeutic outcomes, it is necessary to isolate MSC from a suitable tissue sourcethat posses high neuronal differentiation. In this context, we isolated MSC from extra ocular muscle (EOM) tissue and tested the in vitro neuronal differentiation potential. In the current study, EOM tissue derived MSC were characterized and compared with bone marrow derived MSC. We found that EOM derived MSC proliferated as a monolayer and showed similarities in morphology, growth properties and cell surface marker expression with bone marrow derived MSC and expressed high levels of NES, OCT4, NANOG and SOX2 in its undifferentiated state. They also expressed embryonic cell surface marker SSEA4 and their intracellular mitochondrial distribution pattern was similar to that of multipotent stem cells. Although EOM derived MSC differentiated readily into adipocytes, osteocytes and chondrocytes, they differentiated more efficiently into neuroectodermal cells. The differentiation into neuroectodermal cellswas confirmed by the expression of neuronal markers NGFR and MAP2B. Thus, EOM derived MSC might be good candidates for stem cell based therapies for treating neurodegenerative diseases.


Introduction
Adult stem cells are used extensively for tissue regeneration, repair and also used successfully in several instances to correct genetic disorders in patients [1][2][3][4]. In addition to detailed characterization of the nature of these adult stem cells, there is also a need to identify novel tissue sources from where stem cells could be isolated and manipulated for therapeutic purposes. Adult stem cells from different sources do not differentiate equally into all lineages unlike embryonic stem cells [5]. The differentiation potential of adult stem cells have been closely related to their tissue of origin [6] eventhough they could be induced to trans-differentiate into cells of different germ layer in the presence of induction factors. Mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord blood could differentiate into several mesenchymal as well as non-mesenchymal lineage cell types [7]. These cells have been converted into adipogenic, osteogenic and chondrogenic lineage cells with relatively high efficiency and they functioned and repaired effectively in vivo as well [7]. One of the major areas where cell therapy is much sought after is neuronal repair for spinal cord injury and neurodegenerative diseases. One of the drawbacks associated with using embryonic or tissue specific adult stem cells for neuronal repair is its conversion into cells of redundant lineages in vivo. However, adult multipotent cells which could rapidly differentiate into neuronal cells but show reduced differentiation into other cell types would be an advantage in this scenario.
In the current study, stem cells were isolated from extra ocular muscle tissue and their multipotent differentiation ability was analyzed. Extra ocular muscles (EOM) are a group of highly specialized muscle type that control the eye movements and are distinct from the other skeletal muscles [8] and possess a unique quality of being spared in muscular dystrophies [9,10]. EOM tissue is excised out during corrective surgery for strabismus when a resection (strengthening) is performed. Recent evidences suggest that EOM possesses a high number of stem cells compared to the limb muscles and have highproliferative capacity [11]. The stem cell compartment of EOM cells were determined by their ability to exclude the dye Hoechst 33342 to give rise to side-population (SP) cells [12]. EOM derived SP cells alsoshowed high hematopoietic regenerative capacity after in vivo transplantation [13].
We hypothesized that since EOM tissue is distinct from other tissue types, and highly innervated unlike skeletal muscle, these cells might posses a superior neuronal differentiation capacity. To test this hypothesis, we first studied the growth, differentiation potential and gene expression profiles of EOM derived stem cells and compared them with the bone marrow derived MSC which have multi-lineage differentiation capacity. In the current study, for the first time, we identified MSC from EOM tissue that shared gene expression and phenotype profiles with bone marrow derived MSC. They also differentiated into mesodermal, neuroectodermal cells and indicate a novel source of cells for regenerative therapy.

Materials and Methods
The current study was reviewed and approved by Institute Human Ethics Committee (IHEC) of Indian Institute of Technology Guwahati (IITG).
Sankaradeva Nethralaya Hospital after written informed consent and in accordance with the hospital human ethics committee guidelines. The tissues were collected in vials containing DMEM with antibiotics and processed within 12 hours. The tissue was rinsed briefly in PBS containing 2x antibiotics, mechanically dissociated with forceps and plated in DMEM containing 10% FBS. Fresh media was added regularly until colonies with spindle shaped cells were obtained.

Bone Marrow Mesenchymal Stem Cells
Bone marrow samples were obtained from patients referred to Hematology department of Gauhati Medical College Hospital (GMCH) after written informed consent following GMCH human ethical committee guidelines. Bone marrow cells after red cell lysis were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at a cell density of 1x10 5 cells/cm 2 . Complete media change was performed after 48 hours to remove the non-adherent cells and spindle shaped adherent colonies appeared after 2-3 weeks in culture.

Field Emission Scanning Electron Microscope (FESEM) Analysis
Cells were grown on fibronectin coated coverslips, fixed with ice-cold acetone:methanol (1:1) solution and dehydrated with graded series of ethanol (50%, 70%, 90% and 100%). The cells were gold coated with a sputter coater and viewed under Field Emission Scanning Electron Microscope (Zeiss, Germany).

Immunocytochemical Staining
Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were permeabilised with 0.1% triton X-100 for 20 minutes, washed and stained with primary antibody in 2% FBS solution at 4°C overnight and with fluorescently conjugated secondary antibody for 1-2hr at room temperature. Nucleus was stained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, 5mg/ml) and cells were washed, mounted and documented microscopically (Zeiss, Axio Observer Z1).

Phenotyping by Flow Cytometry
Adherent cells were trypsinized and stained with fluorescent dye conjugated monoclonal antibodies against human cell surface antigens (BD Biosciences). The cells were incubated at 4°C for 30 minutes, washed and analyzed with FACS calibur (Becton Dickinson). Propidium iodide was added before analysis for live/dead separation.
For intracellular markers, the cells were trypsinized and fixed with paraformaldehyde (4%) and permeabilised with TritonX-100 (0.1%) and stained with fluorescently conjugated antibodies for 1hr at room temperature.

SSEA4 Staining
The cells were cultured on fibronectin coated cover slips for 24-48 hours. The cells were washed with PBS and incubated with anti-SSEA4 (Chemicon) antibody for 1 hour. The cells were washed and stained with fluorescent conjugated secondary antibody. The stained cells were microscopically documented.

Colony Forming Unit Assay
One hundred cells per well were seeded in a 6-well plate and cultured in media containing 20% FBS for 14 days. The cells were fixed with ice-cold methanol and stained with May-Grünwald and Giemsa solution. Colonies with more than 50 cells were counted.

Adipogenic, Osteogenic and Chondrogenic Differentiation
EOM-MSC and BM-MSC were differentiated into adipocytes and osteocytes using specific induction media as reported earlier [14]. Briefly, the cells were cultured in DMEM containing 10% serum supplemented with dexamethasone, isobutylmethylxanthine, indomethacin and insulin for adipogenic differentiation and dexamethasone, β-glycerophosphate, and ascorbic acid for osteogenic differentiation. After 21-28 days, differentiation was analyzed by staining for alkaline phosphatase, alizarin red to detect osteogenic differentiation and oil red-O for analysis of adipogenic differentiation. Expression of OSTEOCALCIN and ADIPOQ in osteo-and adipo-differentiated cells respectively were analyzed by real-time quantitative PCR.
For chondrogenic differentiation, micromass cultures were generated in a 12-well plate according to the manufacturer's instructions (Thermofisher scientific). Media change was performed every three days and the differentiated cells were stained with Safranin O after 21 days. Expression of SOX9 was analysed in chondrogenic differentiated cells by real-time PCR.

Clonal Cell Line Derivation
Clonal cell lines were derived from EOM-MSC through limited dilution by plating one cell per well in a 96 well plate. Presence of one cell/well was confirmed microscopically after few hours and proliferating clones were expanded further.

Mitochondrial Staining
Mitochondria in EOM-MSC were visualized by staining with Tetramethylrhodamine, ethyl ester(TMRE) as described earlier [15]. Briefly, TMRE (100nM) was added to the cells in the culture media and incubated at 37°C for 30 minutes. Stained cells were visualized using Zeiss Axio Observer Z1 inverted microscope (Zeiss, Gottingen, Germany).

Karyotype Analysis
For karyotype analysis, colchicine (0.1μg/ml) was added to the cells in culture for 16 hours. The cells were trypsinized and resuspended in 0.56% KCl solution for 30 minutes at 37°C. The cells were washed twice with glacial acetic acid and methanol mixture (1:3) and dropped into clean wet slides. The slides were dried, chromosomes were stained with Giemsa and documented.

Data Analysis
Flow cytometry data was analysed using FlowJo software (Tree Star Inc). Karyotype figures were analysed using Image J software. Statistical analysis was performed with SPSS software and values of p<0.05 were considered statistically significant. The difference in gene expression between EOM-MSC and BM-MSC was analysed by Mann-Whitney non-parametric variables test. The difference in gene expression between control and osteogenic, adipogenic, chondrogenic and neuronal differentiated EOM-MSC samples were tested using paired samples t-test.

Isolation, Cell Morphology and Growth Properties
Fresh tissue samples of EOM resected from the human eye during corrective strabismus surgery of both male and female patients with a median age of 21 yrs were used in this study. The extra ocular muscle tissues obtained from the patients were of lateral rectus, medial rectus or inferior oblique muscle (Fig 1A). EOM tissues were mechanically dissociated and placed on fibronectin coated dishes and cultured in DMEM containing 10% fetal bovine serum. Within 2 weeks of plating, adherent spindle shaped cells emerged from the tissue and formed fibroblastlike colonies. These cells were harvested and further sub-cultured when they reached 70-80% confluency and were termed as EOM derived mesenchymal stem cells (EOM-MSC). EOM-MSC could be cultured in vitro for several passages without change in morphology, growth properties and karyotype. In the current study, the properties of EOM-MSC were compared with bone marrow derived MSC (BM-MSC) obtained from unrelated bone marrow donors.
Morphologically, the EOM-MSC closely resembled BM-MSC, forming spindle shaped cells and proliferated rapidly (Fig 1B-1D). EOM-MSC showed an average doubling time of 34 hours in culture whereas BM-MSC had a doubling time of 42 hours. By immunocytochemistry analysis of cultured cells, we found that EOM-MSC expressed CD90 and the expression did not vary significantly between the different donors ( Fig 1E).
To  (Fig 2A). The expression levels of cell surface markers in EOM-MSC was comparable to that of BM-MSC, except CD49B which was expressed significantly higher in EOM-MSC compared to BM-MSC (48.93%±24.73% in EOM-MSC versus 1.37%±0.44% in BM-MSC, p = 0.0285). We also identified cell surface expression of stage specific embryonic antigen 4 (SSEA4; 57.3%±2.98) by flow cytometry (Fig 2B). Byimmunocytochemistry, it was observed that SSEA4 was expressed predominantly in the spindle shaped cells (Fig 2C). The cell surface marker expression profile of EOM-MSC did not change significantly between different donors or over different passages (passage 2-8).

Differentiation of EOM-MSC into Mesodermal Lineage
The ability to form a single-cell derived colony or the CFU-F (colony-forming unit-fibroblasts) ability of EOM-MSC was analysed and compared with BM-MSC. The CFU-F ability of EOM-MSC was significantly higher than that of BM-MSC (35.17%±11.05% in EOM-MSC versus 12.76%±1.10% in BM-MSC) implying the high proliferation capacity of EOM-MSC ( Fig 3A). Since EOM-MSC resembled BM-MSC closely in their morphology, phenotype and growth properties, their differentiation ability into adipocytes, osteocytes and chondrocytes was tested. EOM-MSC at early passage was induced to differentiate into adipocytes, osteocytes or chondrocytes by addition of specific induction factors and the differentiation percentage was determined. EOM-MSC readily differentiated into adipocytes, osteocytes and chondrocytes as determined by specific staining to detect the differentiation (Fig 3B-3D) and control uninduced cells did not stain for the any of the differentiation markers (data not shown). Differentiation into adipocytes, osteocytes and chondrocytes was further confirmed by gene expression analysis and we observed a significant upregulation of mRNA levels of ADIPOQ in adipocyte differentiated cells, OSTEOCALCIN in cells differentiated into osteocytes and SOX9 in chondrogenic differentiated cells (Fig 3E-3G). However, we noticed that the differentiation ability of EOM-MSC into either adipocytes, osteocytes or chondrocytes waslower than that seen in BM-MSC (Fig 3B-3G).

Differentiation of EOM-MSC into Neuroectodermal Lineage
Although MSC from different organs share a similar phenotype, it has been proposed that their differentiation ability is influenced by the resident tissue [6] from which they were isolated. Although EOM is of mesodermal origin, it is a high innervated tissue [16], we tested to see if they posses neuroectodermal differentiation potential. EOM-MSC were subjected to neuronal differentiation through an intermediate neurosphere stage or direct conversion. EOM-MSC readily differentiated into neuronal cells, which was evident by the observed change in morphology and expression of neuronal lineage specific genes. A significant upregulation of mRNA for the neural markers Nestin (NES)and β-III-tubulin (TUBB3) was observed in the cells induced with the neuronal media ( Fig 4A). Further, the mRNA for neuronal transcription factors Neuronal differentiation 1 (NEUROD1) and Paired box 6 (PAX6) were upregulated five-fold and 45-fold respectively in the neuronal differentiating cells (Fig 4A). However, the embryonic transcription factors OCT4, SOX2 and NANOG expression was downregulated during neuronal differentiation (S1 Fig). Neuronal differentiation in the cells was also accompanied by decrease in the expression of CD146 (Fig 4B) compared to proliferating control cells (Fig 4C) but no change in CD90 expression was observed (Fig 4D). The neuronal differentiation was further confirmed by increase in Glial fibrillary acidic protein (GFAP) in the cells at neurosphere stage and nerve growth factor receptor (NGFR) and microtubule-associated protein 2B (MAP2B) expression in the differentiated cells by immunocytochemistry analysis (Fig 4E-4G).
To understand neuronal differentiation capacity of EOM-MSC further, single cell derived cell lines were obtained from EOM-MSC (Fig 5A and 5B). The clonally derived cells were expanded in vitro and could be differentiated into neuronal cells. Neuronal differentiation of clonal derived EOM-MSC was confirmed by expression of neural marker NGFR (Fig 5C and 5D).

Gene Expression Analysis
To study EOM-MSC further, we compared different groups of genes expressed in EOM-MSC with BM-MSC. Among the several genes anlaysed, mRNA expression of neuronal marker NES was significantly higher in proliferating EOM-MSC compared to BM-MSC ( Fig 6A). In addition, mRNA of self-renewal related transcription factors OCT4, NANOG and SOX2 were also expressed60-100 fold higher in EOM-MSC thanin BM-MSC ( Fig 6A). However, OCT4 protein expression was not detectable in the cells by immunocytochemistry analysis (S1 Fig). No significant difference in expression levels of apoptosis related genes BAD or inhibitor of apoptosis genes cIAP1 and cIAP2 was found between EOM-MSC and BM-MSC (Fig 6B). Factors such HIF1α, IL6 and TNFα were expressed higher in BM-MSC compared to EOM-MSC. Calcium channel related genes ORAI1, STIM1 and TRPC1 expression was similar in both EOM-MSC and BM-MSC (Fig 6C).

Mitochondrial Distribution and Karyotype
Varum et al reported that the stem cells and differentiated cells exhibited varied mitochondrial distribution pattern inside the cells with stem cells characterized by peri-nuclear distribution, whereas the differentiated cells exhibited uniform cytoplasmic distribution [17]. The mitochondrial distribution in both EOM-MSC and BM-MSC were analyzed by staining with TMRE. A peri-nuclear mitochondrial distribution was observed in both EOM-MSC and BM-MSC (S2 Fig), however, cytoplasmic distribution was also seen in EOM-MSC suggesting a heterogeneous population of cells. Lastly, to determine whether the cells underwent any chromosomal abnormality due to ex vivo expansion that will make these cells unsuitable for therapeutic purposes, karyotyping was performed to assess the chromosome number. All the cultures tested in different passages exhibited normal karyotype with 46 chromosomes without any gross abnormality (S3 Fig). Thus, the adherent cells isolated from EOM tissues were morphologically and functionally comparable to MSC obtained from bone marrow and showed high neuronal differentiation capacity.

Discussion and Conclusion
Several studies have enabled MSC to be used effectively for clinical applications. MSC have largely been shown to be isolated from bone marrow, adipose tissue and umbilical cord blood [7]. Each of these sources produced MSC which have the potential to differentiate into multiple cell types and MSC from ocular limbal stroma were also reported to exhibit multipotent differentiation capacity [18]. We show in our current study, extra ocular muscle (EOM) tissue as a novel tissue source of MSC with high neuronal differentiation capacity that could be used for both autologous and allogenic transplantations. Extra ocular muscles are important for controlling the movement of the eye but are excised and discarded during corrective strabismus surgery [19]and could be used for MSC isolation. Our study shows for the first time that multipotent mesenchymal stem cells are present in the human extra-ocular muscle tissues that could be successfully isolated and cultured without compromising their mesenchymal and non-mesenchymal differentiation ability. By merely plating the mechanically dissociated EOM tissue without any enzymatic treatment, spindle shaped cells migrated out of the tissue and formed adherent colonies with an average of 80% of samples yielding expandable colonies of cells. The cell surface expression of CD90, CD73, CD105, integrins and other markers along with spindle shaped morphology, mesenchymal differentiation capacity suggest that EOM-MSC resemble bone marrow or other mesenchymal tissue derived mesenchymal stem cells [20,21]. Nevertheless, EOM-MSC that we isolated were unlike muscle progenitors as they were negative for CD34, a marker of muscle progenitor cells [22,23]. EOM-MSC also did not resemble fibroadipogenic progenitors (FAP) reported by others as EOM-MSC were also negative for FAP marker PDGFRa [24][25][26].
EOM-MSC fulfills the criteria to be termed as MSC [27], and may be used in regenerative therapy for neurological disorders rather than bone repair as these cells seemed to have high neuronal differentiation capacity but low osteogenic differentiation compared to BM-MSC. Low osteogenic, adipogenic and chondrogenic differentiation ability of these cells could be related to the nature of tissue from which these cells were derived [28] which might be of advantage during cell therapy for neurological disorders. Similar to skin and adipose derived MSC, which have been proposed to be derived from mesoderm [29], EOM-MSC might also have a mesodermal origin. However, we found high expression of NESTIN in EOM-MSC which is not only a marker for neural progenitors but also expressed by neural crest cells [30,31]. In addition, EOM-MSC expressed the cell surface marker SSEA4 which is expressed in high levels by embryonic stem cells. Although EOM-MSC showed higher expression levels of transcription factors OCT4, NANOG and SOX2 at mRNA level compared to BM-MSC, we could not identify OCT4 protein expression. Nevertheless, OCT4, NANOG and SOX2 transcript levels were downregulated during neuronal differentiation as reported also by others [32].
Taken together, we have identified previously unstudied multipotent stem cells from the extraocular muscle tissue that has the signature of mesenchymal stem cells with neuronal differentiation capacity in vitro which make them suitable candidates for use in treatment of neurological disorders.