|Year : 2013 | Volume
| Issue : 2 | Page : 71-77
Methods and applications for mesenchymal stem cells
Eman A Ahmedy1, Samia H Kandel1, Samia H Rizk2, Hala M Gabr2, Khaled A Khalifa1, Samar M Kamal1
1 Department of Clinical Pathology, Faculty of Medicine, El Menoufia University, Menufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Kasr El Eini University, Cairo, Egypt
|Date of Submission||05-Mar-2013|
|Date of Acceptance||07-May-2013|
|Date of Web Publication||31-Jan-2014|
Eman A Ahmedy
Department of Clinical Pathology, Faculty of medicine, El Menoufia University, Yassin Abd El Ghaffar street, Shebin El Kom, 32511 El Menoufia
Source of Support: None, Conflict of Interest: None
The aim of this work is to study neurogenesis using mesenchymal stem cells (MSCs) as a model of stem cells and then follow them as they form neurons.
MSCs are multipotent adult stem cells present in all tissues. They are present in the bone marrow, and can differentiate in vitro into neurons, glial cells, and myofibroblasts. MSCs have been proposed as sources of stem cells for regeneration of the central nervous system. Thus, one of the goals of regenerative medicine is to ameliorate irreversible destruction of brain tissue and spinal cord by harnessing the power of stem cells to initiate neurogenesis in damaged areas of the brain.
Materials and methods
MSCs were cultured from bone marrow aspirate and detected morphologically and by flow cytometric analysis of surface markers CD44 and Oct3/4, then differentiated into neural cells using neural induction media, which consisted of a cocktail of retinoic acid dissolved in DEMSO, recombinant human basic fibroblast growth factor, recombinant human epidermal growth factor, and insulin-like growth factor I, and detected by glial fibrillary acidic protein (GFAP).
The results of this study showed that MSCs could be isolated from the bone marrow and assumed the typical fibroblastoid morphology and reached 80-90% confluence at about 9 days. They expressed CD44 with a mean ± SD of 81.54 ± 11.58 and CD Oct3/4 with a mean ± SD of 56.12 ± 17.37. MSCs showed positive expression for double expression of CD44-OCT3/4, with a mean ± SD of 54.03 ± 17.42. A highly significant statistical correlation (P < 0.001) was found between age and double expression of CD44-OCT3/4. No statistically significant correlation (P > 0.05) was found between MNCs and double expression of CD44-OCT3/4. MSCs induced with neural induction media show morphological changes consistent with neurogenesis as compared with the symmetric morphologies of the uninduced cells, as shown by an inverted microscope. Induced cells showed positive staining with GFAP whereas uninduced cells showed negative staining.
MSCs can be isolated successfully from bone marrow aspirate and can be differentiated into GFAP-positive neural cells.
Keywords: Glial fibrillary acidic protein, mesenchymal stem cells, neurogenesis, retinoic acid
|How to cite this article:|
Ahmedy EA, Kandel SH, Rizk SH, Gabr HM, Khalifa KA, Kamal SM. Methods and applications for mesenchymal stem cells. Menoufia Med J 2013;26:71-7
|How to cite this URL:|
Ahmedy EA, Kandel SH, Rizk SH, Gabr HM, Khalifa KA, Kamal SM. Methods and applications for mesenchymal stem cells. Menoufia Med J [serial online] 2013 [cited 2018 May 24];26:71-7. Available from: http://www.mmj.eg.net/text.asp?2013/26/2/71/126094
| Introduction|| |
Stem cells are cells found in all multicellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types , . Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies  .
Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in all tissues, as part of the perivascular population. As multipotent cells, MSCs can differentiate into different tissues originating from mesoderm ranging from bone and cartilage to cardiac muscle  . These cells are present in the adult bone marrow, and can differentiate in vitro into neurons, glial cells, and myofibroblasts  .
Neural stem cells (NSCs) are considered adult stem cells because they are limited in their capability to differentiate. NSCs are generated throughout an adult's life by the process of neurogenesis  . As neurons do not divide within the central nervous system, NSCs can be differentiated to replace lost or injured neurons or in many cases even glial cells  .
MSCs have been proposed as potential sources of stem cells for regeneration of the central nervous system  . Thus, one of the goals of regenerative medicine is to ameliorate irreversible destruction of brain tissue and spinal cord by harnessing the power of stem cells to initiate neurogenesis in damaged areas of the brain  .
It is well accepted that transplantation of MSC, particularly those derived from the bone marrow, promotes tissue repair through secreted soluble factors that enhance tissue regeneration, stimulate proliferation, migration, and differentiation of endogenous stem-like progenitors found in most tissues, as well as by decreasing inflammatory and immune reactions and apoptosis  . The ability of such cells to modify the tissue microenvironment through its trophic influence may contribute more significantly than their capacity for transdifferentiation in effecting tissue repair  .
| Materials and methods|| |
Calcium-free and magnesium-free PBS was purchased from Invitrogen (Faraday Ave Carlsbad, USA), Ficoll-Hypaque solution (density 1.077 g/l) was purchased from Biochrom AG (Leonorenstr, Berlin), sterile cell culture low-glucose (1000 mg/l)-Dulbecco's modified Eagle's medium (DMEM) with l-glutamine (2 mmol/l), and fetal bovine serum (FBS) were purchased from Euroclone (Via Lombardia, Siziano, Italy). Penicillin/streptomycin, trypan blue 0.04%, and retinoic acid 20 mmol were purchased from Sigma (Saint Louis, Missouri, USA), trypsin EDTA 0.25% was purchased from Lonza (Verviers, Belgium), and Fungizone (0.25 μg/ml) was purchased from Bioscience (Redhill, Surrey, United Kingdom). Dimethylsulfoxide was purchased from MERCK (Darmstadt, Germany). Bovine fibronectin was purchased from the R&D system (McKinley Place NE, Minneapolis, USA) and fixation and permebilization reagent was purchased from Beckman Coulter (Avenue de Lattre de Tassigny, Marseille, France).
Antibodies and cytokines
Fluorescein isothiocyanate anti-human CD44, phycoerythrin anti-human CD OCT3/4, recombinant human basic fibroblast growth factor (b-FGF), b-FGF and basic epidermal growth factor, and insulin growth factor 1 were purchased from R&D system.
Culture of MSCs
The present study included25 patients between 18 and 50 years of age. It was carried out in the Clinical Pathology Department, Kasr El Eini Medical School, Cairo University, and El-Monofiya University Hospital. An extra bone marrow sample was aseptically collected from patients who already have another indication for bone marrow examination after obtaining their consent. Bone marrow aspirate (10 ml) was diluted with sterile PBS in the ratio of 1 : 1 and layered on top of Hicoll-Hypaque. The MNC fraction was collected and seeded at a concentration of (million cells/cm 2 ) and allowed to adhere to tissue culture plastic flasks 25 cm 2 (cell star; Maybachstrasse, Frickenhausen, Germany), incubated at 37 C and 5% CO 2 in 5 ml of the fresh complete nutrient medium (F10), which included the following: low-glucose DMEM with l-glutamine (2 mmol/l), 10% FBS, penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin), and Fungizone (50 μl/ml) (0.25 μg/ml). Half medium was changed every 4 days to remove nonadherent cells.
At day 9, when fibroblast-like cells reached 80-90% confluence, these cells were harvested by trypsinization. The harvested cells were examined morphologically and by flow cytometric analysis of surface markers CD44 and OCT3/4.
MSCs were plated on sterile cover slips in 35-mm suspension tissue culture dishes at a density of 500-1000 cells/dish in neural induction media (NIM). Retinoic acid (RA) was added to F10 medium at 30-μmol/l final concentration and recombinant human b-FGF 10 ng/ml and recombinant human epidermal growth factor (rHEGF) at 10 ng/ml. Insulin-like growth factor I 10-25 ng/ml was then added. After 3-4 days, medium was replaced with fresh medium. At different times after induction, cells were studied by morphology, immunofluorescence.
GFAP immunofluorescence staining of plates
On the fifth day, a sterile fibronectin-coated cover slip was inserted into 35-mm suspension tissue culture dishes. Cover slips were seeded with 500 cells in NIM. The cells were fixed with 0.5 ml of 4% paraformaldehyde in PBS for 20 min at room temperature, then washed, permeabilized, and blocked with 0.5 ml of 0.3% Triton X-100 and 1% FBS serum in PBS at room temperature for 45 min. After blocking, the cells were incubated with 300 μl/plate of 1× mouse anti-human glial fibrillary acidic protein (GFAP) overnight at 2-8°C in the dark, then washed and incubated with 300 μl/well of secondary antibody in the dark at room temperature for 60 min. After washing, they were immediately examined using a fluorescent microscope.
| Results|| |
The average age of the patients ranged from 18 to 50 years, with a mean ± SD of 34.16 ± 10.31 [Table 1]. The average number of MNCs (million) ranged from 45 to 80 million, with a mean ± SD of 61.04 ± 11.7. The viability ranged between 95 and 98%. No statistically significant correlation (P > 0.05) was found between age and mononuclear layer.
|Table 1: Descriptive statistics of age and MNCs count among the studied group|
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Examination of the MSCs
Morphological examination of MSCs
At days 1 and 2 after the initial plating, the adherent cells showed cytoplasmic projections and a tendency to form small clusters [Figure 1]. The onset of fibroblast-like cell formation could be observed approximately at days 3 and 4 [Figure 2]. MSCs grew as a monolayer of large, flat cells. At day 5, cells tended to assume a spindle-shaped morphology. At day 6, spindle-shaped cells increased in number and tended to form colony-forming unit fibroblast that were cell clusters of more than 30 cells originating from one clonal cell  . At day 7, cells showed multipolar fibroblastoid cells and 60% confluence that gradually increased to reach 80-90% confluence at about 9 days [Figure 3].
Identification of MSCs by flow cytometry
MSCs showed positive expression for CD44 (ranging between 60.30 and 98.9, with a mean ± SD of 81.54 ± 11.58). MSCs showed positive expression for CD Oct3/4 (ranging between 29.5 and 89.8, with a mean ± SD of 56.12 ± 17.37). MSCs showed positive expression for double expression of CD44-OCT3/4 (ranging between 27 and 88.3, with a mean ± SD of 54.03 ± 17.42) [Table 2]. A highly significant statistical correlation (P < 0.001) was found between age and double expression of CD44-OCT3/4 [Table 3]. No significant statistical correlation (P > 0.05) was found between MNCs and double expression of CD44-OCT3/4 [Table 4].
|Table 2: Descriptive statistics of flow cytometric results CD44, CD OCT3/4, and double expression of CD44-OCT3/4 (%) on MSCs|
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|Table 3 Pearson linear correlation between age and double expression of CD44-OCT3/4|
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|Table 4 Pearson linear correlation between MNCs and double expression of CD44-OCT3/4|
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Characterization of neuronal cells transformed from BMA-derived MSCs
Postinduction morphological changes
MSCs induced with NIM show morphological changes consistent with neurogenesis as compared with the symmetric morphologies of the uninduced cells, as shown by an inverted microscope. The cells progressively showed a typical neuronal perikaryal appearance. The region around the nucleus became narrower and thicker, to form a cell body-like structure named refractive somas, whereas the rest of cytoplasm was elongated to give rise to multiple cellular processes [Figure 4]. At the eighth day after induction, the cells contracted and became smaller, and formed bipolar or multipolar prominences extending from the cell body [Figure 5]. A single, long axon-like process also developed. Some neurons showed pyramidal cell morphologies. Neural morphology was distinguished from the normal cell morphology by a number of criteria, including the refractile appearance of the cells, the presence of either bipolar or multipolar neurite-like projections (with the projection at least equal to or exceeding the diameter of the soma), and the occasional presence of secondary extensions [Figure 6].
GFAP detection by immunofluorescence
Induced cells showed positive staining with GFAP whereas uninduced cells showed negative staining [Figure 7].
| Discussion|| |
MSCs are found in different tissues such as bone marrow, fat, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovial membrane, and skeletal muscle  .
NSC transplantation has been proposed as a future therapy for neurodegenerative disorders. However, NSC transplantation is hampered by the limited number of brain donors and the toxicity of immunosuppressive regimens that might be needed with allogeneic transplantation. These limitations may be avoided if NSCs can be generated from clinically accessible sources, such as bone marrow and peripheral blood samples, that are suitable for autologous transplantation  .
Recently, it had been proved that MSCs aid the stimulation of angiogenesis, neurogenesis, and regeneration of damaged tissue  .
Bone marrow MSCs had also shown promise in in-vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease  . Although bone marrow cellularity is expected to decrease with age because of a decrease in the hematopoietic compartment as a result of variation in the composition of the stromal cell microenvironment  , no significant statistical correlation was observed between age and mononuclear cell number in the bone marrow in the present study. This might be attributed to the nature of the bone marrow samples used, donated mainly by patients with hyperactive marrow conditions with increased cellularity (e.g. ITP, hypersplenism, etc.)  .
Whereas hematopoietic stem cells constitute about 1% of the bone marrow population, MSCs constitute only 1/10 000-1/100 000 of the bone marrow nuclear cells  , a fact that necessitates their expansion in vitro before use. Low-glucose DMEM (1000 mg/l) was used in this study because MSCs utilize energy more efficiently under restricted glucose treatment and show greater self-renewal and antisenescence abilities, whereas their differentiation potentials remain unaffected  .
In the present study, MSCs were detected by their morphology and confirmed by its surface marker as CD44, which was positive on isolated MSCs, ranging from 60.30 to 98.9%.These data were in accordance with Orcaina and colleagues , . However, Zvaifler et al.  reported it as a negative MSC marker. Moreover, Fu et al.  had isolated cells that expressed the characteristic antigens of MSCs, including CD29, CD44, CD73, CD90, CD105, and CD166, and did not express hematopoietic markers CD45, CD34, CD14, or CD11. Lin et al.  had also shown positive CD44 and negative CD34, working under the same conditions as ours. CD44 acts as a 'bone homing receptor', directing migration of human hematopoietic stem cells and MSCs to the bone marrow  . CD44 glycosylation directly controls its binding capacity to fibrin and immobilized fibrinogen  .
There has been a common opinion that CD73, CD105, CD90, and CD44 are highly specific for MSCs, and hence can discriminate multipotential cells from the tissue resident fibroblasts. However, several studies showed that these markers were ubiquitously expressed on stromal cells from many locations as well as on skin fibroblasts  and at best they only inform an investigator that the phenotyped cells are nonhematopoietic and stromal in origin  .
Oct3/4 in the present study was positive in the range of 29.5-89.8%. This was in agreement with Miura et al.  , who also reported it as a positive MSCs marker. In adult stem cells, several studies suggest a role for Oct-4 in sustaining the self-renewal capacity of adult somatic stem cells (i.e. stem cells from epithelium, bone marrow, liver, etc.)  .
In the present study, double expression of CD44 and OCT3/4 ranged from 27 to 88.3%, indicating that a high percentage of mesenchymal cells also show a preserved stemness nature. These findings were not in agreement with those of Orciani et al.  , who succeeded in isolating MSCs with positive double-expressed CD44 and OCT3/4 surface marker. However, a highly statistically significant negative correlation was observed between age and double-expressed CD44-OCT3/4, indicating an actual decrease in the number of MSCs with age. These findings were in agreement with those of Stolzing et al.  . In contrast, Payne et al.  reported no correlation of MSC number with donor age; this may be attributed to their different protocol of work, for example, they obtained their bone marrow samples from the femur. In the present study, differentiation was achieved using a cocktail of RA and growth factors. Khoo et al.  reported that growth factor-based neural differentiation has yielded promising results. In contrast, the use of a chemical-based method in culture had been controversial because of the toxic effect of these chemicals in modifying cell size and shape  . RA A is an active derivative of vitamin A. Its wide expression in the nervous system during development, its role as a potent inducer of cell differentiation, and the wide expression of its receptors and binding proteins in the brain suggest an important role in brain development and function  . This was in agreement with Montiel-Eulefi et al.  , who succeeded in generating neural cells using RA and other neural inducing agents.
In the current study, postinduction cells showed positive staining with GFAP on the fifth day after induction whereas preinduction cells showed no staining. These findings exclude the use of neural committed cells from the bone marrow sample.
These findings were in contrast to those of Sanchez-Ramos  , who had first described neural differentiation by MSCs and detection of nestin and GFAP after differentiation, but, importantly, failed to report the pre-differentiated phenotype of the cells.
Reyes et al.  had succeeded in culturing GFAP-positive nerve cells that also expressed tubulin III, NSE, glutamate Gal-C, MAP (other markers of nerve cells) from human bone marrow mesenchymal stem cells (BMSC) by plastic adherence cultured in low glucose, DMEM, FBS, EGF, and PDGF.
Sanchez-Ramos had reported that co-culturing of BMSC with fetal mouse midbrain significantly increased the percentage of BMSC that expressed NeuN and GFAP (markers of neurons and astroglia, respectively). The co-culture experiments support the hypothesis that cell-cell contact, in addition to signaling with trophic factors and cytokines, plays an important role in the differentiation of these BMSC  .
Fu et al.  have observed astrocytes that were GFAP+ and/or S-100μ+ using a cocktail of 20 ng/ml of both EGF and b-FGF. However, the protocols of Kohyama et al.  failed to differentiate MSCs into glial cells because no GFAP-immunoreactive cells were found.
| Conclusion|| |
MSCs can be isolated successfully from bone marrow aspirate samples. The bone marrow should be considered a very valuable source of MSCs. Bone marrow MSCs can differentiate into GFAP-positive neurons under the effect of a cocktail of RA, recombinant human b-FGF, rHEGF, and insulin-like growth factor I.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]