ARC Journal of Neuroscience

AUTHOR DETAILS

Rafal H. Abdullah

Iraqi center for cancer and medical genetic research/Al-mustanserya university
Baghdad –Iraq
[email protected]

Nahiyosefyaseen
Iraqi center for cancer and medical genetic research/Al-mustanserya university
Baghdad –Iraq
[email protected]

Shahlla M. Saleh Al-Nahrain University
[email protected]

Ahmed MajeedAlshamary
Iraqi center for cancer and medical genetic research/Al-mustanserya university ahmed
[email protected]

KEYWORDS

mesenchymal stem cell, motor neuron, acetylcholine.

INTRODUCTION

Spinal cord injury is a lesion of traumatic nature result to disruption of nerve fiber bundles effect on the ascending sensory and descending motor information(Julio,et.al,2012) .Stem cell represents a promising way for treatment of this injury because self-renewal and their ability to become any cell in an organism like neuron and glia cell. Neuroprotective and axon regeneration promoting effects have also been credited to transplanted stem cells (Tewarieet al., 2009). Mesenchymal stem cell treatment for spinal cord injury improves functional recovery and enhanced astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplantation of mesenchymal stem cells is limited by poor survival of the cells in the damaged nervous tissue (Ritfeld and Oudega , 2014; Cui et al., 2014).

Motor neurons are specialized nerve cells in the brain and spinal cord that transmit the electrical signals to muscle and generate movement. There are two group of motor neuron, the upper motor neuron at the top of the brain in motor cortex and extended down in the spinal cord to connect the lower motor neurons which travel out of the spinal cord and connect the muscle (Talbot, 2008).

Microtubule associated protein 2 (MAP-2) and Acetyl choline transferaseare differentiation markers for motor neuron. High-performance liquid chromatography (is a technique in analytical chemistry used to separate the components in a mixture, to identify each component, and to quantify each component (Kupiec, 2004). Therefore Acetyle choline concentration detected in culture media which is a neurotransmitter at neuromuscular junctions responsible for synapses in the ganglia of the visceral motor system, and at a variety of sites within the central nervous system (Purves et al.,2001).

The development of 3D scaffolds, support structures or matrices that induce cells to form functional tissues, is one of the main object of tissue engineering and regenerative medicine research (Langer and Vacanti, 1993).

Collagen type I used to form three dimensional cell culture matrix composed of collagen fibres are able to support short- and long-term growth of various cell types, including cancer cell lines, endothelial cells, endometrial cells, hepatocytes, osteoblasts and fibroblasts and to sustain or even enhance cell differentiation, in vitro, 3-D culture systems have been successfully used in the investigation of complex biological processes, such as angiogenesis, wound healing, tumour invasion , metastasisand simulate cell-cell interactions, which take place in tissues under physiological and pathophysiological conditions (Themistocleouset al., 2004).

Mesenchymal stem cell differentiation into functionally active motor neuron cell using Retinoic acid and Sonic hedgehog growth factor then studying elongating capability of differentiated cell axon and mimicking the normal tissue structure by in collagen matrix.

MATERIAL AND METHODS

Mesenchymal Stem Cell Isolation

Bone marrow mesenchymal stem cell isolated from thigh of Albino mice used ,in using flushing method was used by flush the bone marrow cells from bones using a 1ml syringe containing 1ml of growth culture media (MEM). freshly isolated whole bone marrow cells were resuspended in 5ml growth culture medium MEM supplemented with 20 % FBS, 1 % Ampicllin/Streptomycin in 37C (Soleimani, M.and Nadri,2009).when the cultures reached 80 - 100 % confluence ,the cells were suspended in culture at density (>106 cells /ml). the cell viability studied by using trypan blue dye which allows distinguishing between healthy cells with uncompromised membrane integrity (unstained) and dead ones (stained blue).The Immunocytochemistry analysis of MSCs was done by CD 105 CD90+, CD 34¯, CD 44+ (Lin,et.al,2013).

Differentiation of MSC into Motor Neuron Cell

In motor neuron differentiation the 1st passage of (2 ×104) of MSCs were used. The differentiation strategy involved two main steps (preinduction step) the cells were cultured in a MEM medium supplemented with 20% FBS and 1 m MB etamercaptoethanol (Santa Cruz biotechnology). After 24 h of incubation the media was discarded and MEM free serum media containing 2mM BME was added and incubated for 1h. Then media was discarded and cell washed with free serum media. At the second step (induction step) which lasted for four days MEM free serum media with 1mM retinoic acid(Santa Cruz biotechnology), 10ng/ml nerve growth factor(Santa Cruz biotechnology)and 0.1 ng/ml sonic hedgehog (Santa Cruz biotechnology )were added and incubated at 37℃ for 4 days. As a negative control, MSCs were cultured in medium without differentiation stimuli along with the differentiation experiments in the same conditions. Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37℃. Cultures were maintained by medium exchange every 2 days. The cell morphology was observed under inverted microscope according to modification method of (Hu and Zhang, 2009).

Motor Neuron Detection by Immunocytochemisty

After that the media was aspirated and the cell was fixed by 4% paraformaldehyde for 10 min, incubated with 1% hydrogen peroxide for 10-15 minutes and wash with PBS for 5 min three times.Aliquot of 1.5% blocking serum was added to cell section for one. Cells sections were incubated with diluted acetyletransferase antibody at a ratio ( 1:50 vol/vol) overnight as production company instruction and diluted microtubule associated protein antibody at a ratio (1:100vol/vol) for 1 h then washed. Cell sections were incubated for 30 min with 1.2 ml biotinylated secondary antibody.A liquid of 650 μl of AB enzyme reagent was added to cell section and washed two times with PBS for 2 min. Three drops of peroxidase substrate was added to cells for 10min, or until desired stain intensity develops and washed. Hematoxylin stain was added to cell section for 5-10 seconds and immediately washed with distilled water. Finally 1-2drops of permanent mounting medium was added and examined by light microscopy at magnification powers 200X and 400X (Moral-Sanz, et.al. 2012).

Scanning by Electron Microscope

Scanning electron microscope solutions (4% gluteraldehyde). It was prepared by mixing 4 ml of stock gluteraldehydewith 94 ml D.W. .It was prepared by dissolving 1mg of osmium tetroxide in 10ml D.W., Thenstored in dark container at -20.The slides were washed by PB Sand fixed with gluteraldehyde 4%for 24h at 40C then Osmium solution was added for 2 h in dark. At the end of two hour, slides were washed with PBS two times. A serial dehydration for the sections were carried out started from (50, 70, 95,and 100) % for 30 min,dried and examined under the electron microscope (Eisenbach,2015).

Motor Neuron Function Detection

The activity of motor neuron detected using HPLC by estimation acetylcholine concentration in media at different period of differentiation. The test was done in IbinSenaCenter /Ministry of higher education and scientific research, usingshimachzu lc-2012 AHT machine.

HPLC Conditions
Standered Acetylcholine was prepared by dissolving 1 mg acetylcholine chloride powder >98% purity in 1ml D.W.

A column 250*4.6 mm colume 5micron lava with Injection volume 0.7 ml/min. Column temperature was18 °C, Detection wavelength: 210 nm and Mobile phase: Acetone, water , methanol 80%, 14%, 6%(Landgraf et .al,2010) .

Acetylcholine concentration was calculated according to this equation (Chen and Yun, 2009).

Concentration of compound (ppm) = (area of sample × conc. of standard)/area of standard)

Assay of Elongating Moto Neuron Cell Axon

Mesenchymal stem cell was cultured in five tissue culture flask at 37oC in CO2 incubator until monolayer formed. Media was discarded and pre induction media was added to each flask .At the end of pre induction time, the preinduction media was discarded and induction media containing different concentrations of nerve growth factor started from 10 ,20,40,80 to160 ng/ml (Lentz,etal. 1999).

Axon Length Measurement Program

The axon length was measured using mini divi camera picture program measurement (Qiang et al., 2010; Olsson-Carter et al., 2010 and Cregg et al., 2010).

Three Dimension Collagen Culture

Preparation of neutralized collagen solution

Neutralized collagen solution was prepared by mixing 8 parts of chilled collagen solution to 1 part 0.01M NaOH and 1 part 10X buffered serum free cell cultured medium. Then pH was adjusted to 7.4±0.2 and poured on 3D seeding plate with 8 wells (the thickness of collagen gel should be 1-2mm) and incubated at 37 0C for 20 min. The plate left over night to dry and rinsed with DDW to remove excess salt and to rehydrate collagen gel and used immediately or dried again and stored for weeks at 2-8 C as manufacturer instruction (Wuensch, et.al.2013).

Three Dimensional Culturing

Mesenchymal stem cell was cultured on neutralized collagen seeding plate with 8 wells according to Artym and Matsumoto, (2010) with modification 6X106 cell\20 ml of 20% FCS culture media, then 400μl of cell suspension was added to each well of the neutralized collagen seeding plate and incubated in CO2 incubator at 370C until monolayer formed. Every 2-3 day, slide media was aspirated carefully without contact to the collagen to prevent its shrinking or damage from the side wall of slide and new culture media added carefully. Motor neuron differentiation was induced as previous.

At the end of differentiation motor neuron detection by immunocytochemistry was carried out as mentioned above.

RESULTS

Motor Neuron Induction

Mesenchymal stem cell have the ability to differentiated into neuron like cell and more specialized cell like motor neuron when using simple differentiation factors in practically short period of time not more than 5 days.

There are two stages of differentiation using monolayer of passage one MSCs, The first stage of differentiation, a pre induction stage for 24 h showed that the fibroblast like cell MSC cell membrane withdraw to the middle of the cell, extension became appear and the cell still viable with account 1.9 X106 and attached to the flask surface Figure (1). The media discarded and incubated for 1hour in 2mM β ME of free serum MEM, the cell became more radical in shape, multi polar, and extension increased in number of viable count to about 1.40 X106.

figure1

The second stage of differentiation was the induction stage in which used a combination of Retinoic acid and a Sonic hedgehog factors for just four days, cell body became typical like motor neuron cell, shiny nuclease and with the cell soma have many dendrites and long axon like structure ended with small extensions. The MSCs under three thousand increase size using scanning microscope appeared as circular to spindle shape with no extended extension from the cell while the differentiated cell into motor neuron showed cell body contain dendrites and long extended axon ended with small dendrite under two thousand increase in size Figure (2) and (3).

figure2 figure3

Motor Neuron Cell Detection using Immunocytochemistry

Bone marrow mesenchymal stem cell differentiation into motor neuron cell was detected by immunocytochemistry study. Motor neuron cell are mature differentiated cell have microtubule associated protein 2 (MAP2) and cholinacetyltransferase (CHAT) as specific marker. Results revealed that 90% of differentiated motor neuron was positive for MAP2 and about 85 % for CHAT Figure (4).

figure4

Motor Neuron Function Detection

The activity of motor neuron by measuring the level of acetylcholine produced by differentiated cells in culture media. The increase level of neurotransmitter acetyl cholineproduced in culture media estimated functionality of motor neuron.

Results revealed that a significant increase (75.0±0.57pmm) in Acetyl CoA produced by motor neuron in differentiation medium at 4th day after induction stage in comparison with zero day and after 24h (15.43±0.38 and 15.36±0.44pmm) Figure (5).

Motorneuron cell produce acetyl choline as cholinergic compound from choline and acetyl co A metabolized by enzyme Acetyl choline transferase which expressed strongly in differentiated cell immuonocytochemicaly by CHAT primary antibody as shown in figure (5), therefore acetylcholine present in media indicate that motoer neuron functionally active cell .

figure5

A Assay of Elongating Moto Neuron Cell Axon

Results showed the highest elongation of motor neuron cell axon with a significant increase was (1.93 ±0.185) mm noticed at a concentration 160 ng/ when a nerve growth factor was added to motor neuron differentiation media in the induction stage in addition to RA and SHH compared with control Figure (9-3 A) . Results showed a significant increase in axon length was observed (0.833± 0.145, 0.86 ±0.145, 1.3± 0.10 and 1.76 ±0.317 vs 0.433± 0.145) mm respectively in comparison with control when serial concentrations of NGF added to culture media (10, 20, 40 and 80) ng/ml respectively Figure(9-3) and (10-3). This indicates that NGF induced axon elongation when added to the differentiation culture media into motor neuron cell. Nerve growth factor have an elongation capability for motor neuron cell axon with increasing concentration represent a promising tools for producing spinal cord motor neuron cell with long axon.

figure6 figure7

Differentiation of Mesenchymal Stem Cell in Three Dimensional Cultures

Neutralized collagen matrix in 8well tissue culture slide had a smooth surface area as shown in (A and B) Figure (8) then mouse bone marrow MSCs cultured on the surface of matrix using the previous differentiation media for the same period of time successfully to mimic in vivo structure of brain and spinal code tissue. The cell and matrix after fixation and staining showed like thin lens layer as shown in in Figure (8).

Mesenchymal stem cells in collagen matrix in CO2 incubator showed either spindle shape with two or three polar cytoplasmic processes and extended within matrix or semicircular structure on three-dimensional culture conditions and accumulated together on matrix and some are overlaped Figure(8)

The pre induction stage of differentiation of MSCs into motor neuron cell on collagen matrix in CO2 incubator showed that the cells became penetrating in matrix and had more cytoplasmic extend forming neural like structure. At the end of the induction stage the cell had big soma structure contain many extensions of dendrites and in the other side long extended axis like axon ended with small dendrites like in structure the motor neuron cell Figure (9).

Stem cells differentiated on Hydrogel with culture media contain 1μM RA added regulated formation of neural and astrocyte lineages and when RA and Shh were added to the culture, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation.

figure8 figure9

Results revealed that differentiated motor neuron on collagen were positive for MAP2 and for CHAT Figure (9)

figure10 figure11

Motor neuron cells differentiated from MSC in this study showed positive results for the differentiation marker MAP-2 and CHAT when detected immuonocytochemicaly on the surface of the matrix, Figure (12).

figure12

DISCUSSION

Motor Neuron Induction

Scientists works on bone marrow culture in 1mM BME showed that cell cytoplasm retract and when exposed to 5mM BME for 6 hours, the cell had multipolar body structure (liu,et al., 2011) and also cells incubated in BME for few hours were elongated and beard process like extensions and were immunoactive for neuron specific enolase, nestin, glial fibrillaey acidic protein (Mareschiet al, 2006) and this is as well showed in the results and picture .

The first and second stages of induction represent the most effective, efficient and shortest induction time method for differentiation of mouse bone marrow MSC into motor neuron cells in compare with Shetty and his workers (2015) whose cultured cells in complicated culture media as pre induction, induction in RA and forskolin for 24 h and finally 6-8 days in RA and SHH but they started culturing forming neurospher from MSC on unattached surface flask.

Other workers induce differentiation of MSCs into MNCs for longer time up to two weeks using large amount of growth factor and specific culture conditions like neural body induction and then differentiation (Ebrahimin- Barough, et al .2014).

Motor Neuron Induction

Scientists works on bone marrow culture in 1mM BME showed that cell cytoplasm retract and when exposed to 5mM BME for 6 hours, the cell had multipolar body structure (liu,et al., 2011) and also cells incubated in BME for few hours were elongated and beard process like extensions and were immunoactive for neuron specific enolase, nestin, glial fibrillaey acidic protein (Mareschiet al, 2006) and this is as well showed in the results and picture .

The first and second stages of induction represent the most effective, efficient and shortest induction time method for differentiation of mouse bone marrow MSC into motor neuron cells in compare with Shetty and his workers (2015) whose cultured cells in complicated culture media as pre induction, induction in RA and forskolin for 24 h and finally 6-8 days in RA and SHH but they started culturing forming neurospher from MSC on unattached surface flask.

Other workers induce differentiation of MSCs into MNCs for longer time up to two weeks using large amount of growth factor and specific culture conditions like neural body induction and then differentiation (Ebrahimin- Barough, et al .2014).

Motor Neuron Cell Detection using Immunocytochemistry

Differentiation indication markers MAP-2 used in this study and the results proved that mature spinal cord neuron, MAP-2 a cytoskeletal protein, present in the axon and dendrite of the cell body reveled by mRNA in situ hybridization (Hirokawaet al.,1996).

Bi, et al.(2010). found that the cells have three neural related marker expression increased nestin, NES and MAP-2 with increasing concentration of Retinoic acid by Real time PCR, these factor determined the differentiation into neuron.

The other marker improved the differentiation into motor neuron cells is acetyle choline transferase (CHAT), the enzyme responsible for biosynthesis of acetylcholine and present in the functional cholinergic neurons of the central and peripheral nervous system indicate motor neuron cell (Oda ,1999) and also other studies on embryonic stem cell differentiation into motor neuron using sequence of culturing procedures detected immunocytochemistry using Quantification of HB9 and CHAT cells showed just 30%of total cell are mature motor neuron(Hester, 2011).

Motor Neuron Function Detection

Liu, et al. (2013) showed the production and secretion of acetylcholine from motor neuron cell derived umbilical cord mesenchymal stem cell, positive for acetylcholine transfarase, increased during the period of differentiation from very little in the 9th day of 15 differentiation day, about 2.8 ng/ml to 5.7 ng/ml at the end of differentiation using heparin end fibroblast growth factor comparing with mesenchymal stem cell control which not produce any acetylcholine .

Murine and human embryonic stem cell synthesize acetyl choline and express acetyl choline transferase in regulation of embryonic stem cell as calcium dynamics, cell survival and proliferation,it’s increased the viability, but decreased the proliferation of embryonic stem cells this indicate the presence of low concentration of acetylcholine in the culture of MSCs and pre induction stage of differentiation (Landgrafet .al,2010; Takahashi ,2015)

Assay of Elongating Moto Neuron Cell Axon

Lentz et al. (1999) studied the effect of NGF on the sensory nerve cell axon during the even embryogenesis and showed that different concentration of NGF had different effect on the length of axon in which culture without NGF produce cell with small extended axon. Also a significant increase in axon length were shown in study of Martin et al., (2002) on olfactory neuron axon when 50 ng/ ml of NGF added to culture of cells. One of GTP-superfamily binding protein is inactivating by NGF through prevention of axon extension via the actin cytoskeletal so that differentiation culture media contain NGF increase the axon length (Stocum, 2012).

Differentiation of Mesenchymal Stem Cell in Three Dimensional Cultures

This phenomena showed also in other study of MSCs on collagen gel matrix containing fibronectin. Cells were adjacent in many parts, in direct contact and lined up in a row while and in 3D collagen–laminin gel, cells exhibited a highly elongated star shape and three-dimensional cultured cells appeared to present directional growth, a form of neuritis outgrowth found in neuronal cells (Lee, et al., 2011).

Culturing cells by Artym and Matsumoto (2011) insidethree-dimensional (3D) collagen matrices indicated the increasingly popular and enlarge the understanding of cellular processes and cell - ECM interaction. These indicate that synergistic role of micro environmental cues ESC differentiation and maturation, with potential applications in cell transplantation therapy (Kothapalli and Kamm, 2013).

Other scientific work on MSCs cultured on extracellular matrix and Matrigel immobilize gell showed greater differentiation into neural cells than 2D culture indicating aspecial interaction between cell and extracelullar matrix. Also cells cultured on extracellular-grafted showed higher neural differentiation ratios than those on extracellular 2Dculture this strenght the resulted cells after differentiation with motor neuron structure (Higuchi,et al,2011).

Human embryonic stem cells hEnSCs differentiation into motor neuron using in (polylactic-co-glycolic acid) PLGA showed that PLGA scaffolds enhances more the differentiation of hEnSCs into motor neuron-like cells in comparison with tissue culture plats. So PLGA nanofibers provide a suitable topography for motor neuron differentiation of hEnSCs. The expression of Islet-1 (80%), Chat (89 %), NF-H (50%), and beta-tubulin III (40 %) in PLGA group were higher than the expression of Islet-1 (70 %), Chat (79 %), NF-H (40 %), and beta-tubulin III (40 %) in tissue culture plats (Ebrahimi-Baroughet al.,2014) .

CONCLUSION

Mesenchymal stem cell represent agood source for differentiation into functional motor neuron using less growth factor and time as differentiation of embryonic stem cell and elongation of motor neuron cell axon for future using in spinal cord repair . Collagen matrix represents a benefit structure for miming the normal tissue structure for differentiation of MSC to motor neuron.

Glossary

Mesenchymal stem cell have retracted capacity of differentiation towered few lineage of cell differentiated mainly from mesoderm embryonic germ layer give rise to skeletal and connective tissue.

Motorneuron Motor neurons are specialized nerve cells in the brain and spinal cord that transmit the electrical signals to muscle and generate movement.

Axon elongation an increasing cell axon that extends from the cell body using special growth factor.

Collagen matrix supportive structures that induce cells to form functional tissues, is one of the main object of tissue engineering and regenerative medicine research.

ACKNOWLEDGMENT

I would like to express thanks to the ICCMGR and every single staff member in it, thank you for helping, supporting and kindness.