Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 58, Num. 2, 2010, pp. 201-208

Neurology India, Vol. 58, No. 2, March-April, 2010, pp. 201-208

Original Article

Isolation, characterization and differentiation potential of rat bone marrow stromal cells

Naresh Polisetti¹, VG Chaitanya², Phanithi Prakash Babu², Geeta K Vemuganti¹

¹ Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Hyderabad, India
² Department of Animal Sciences and Biotechnology, University of Hyderabad, Hyderabad, India

Correspondence Address: Geeta K Vemuganti, Head, Ophthalmic Pathology Service, Head, Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad - 500 034, India, geeta@lvpei.org

Date of Acceptance: 23-Oct-2009

Code Number: ni10056

PMID: 20508336

DOI: 10.4103/0028-3886.63789

Abstract

Background: Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages.
Materials and Methods: Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells.
Results: Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, β-tubulin III, neurofilament and synaptophysin.
Conclusion: Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage.

Keywords: Adult stem cells, bone marrow, bone marrow stem cells

Introduction

Bone marrow is a complex tissue containing stem cells with hematopoietic properties. The hematopoietic stem cells, which are the primary source of blood cells in the adult body, are regulated within a microenvironment of stromal cells in the bone marrow. ^{[1],[2],[3],[4],[5],[6],[7]} The stromal cells exert their effects on the hematopoietic cells through direct cell-cell interactions as well as by the release of soluble factors. ^[8],[9],[10] Stromal cells isolated from bone marrow (BMSC) are heterogeneous and fibroblastic in appearance. ^[11] In 1974, Friedenstein et al.^[12] isolated fibroblastoid cells in bone marrow by plastic adherence. Fibroblastoid cells make up 0.001-0.01% of bone marrow cells and display a colony forming unit (CFU-F). They were initially named plastic-adherent cells or colony-forming-unit fibroblasts and subsequently referred to as either marrow stromal cells or mesenchymal stem cells (MSC), due to their potential to differentiate into various connective tissue lineages including adipocytes, osteoblasts, chondrocytes or myoblast. ^[13],[14]

Bone marrow derived MSCs have been isolated from a variety of species, including mouse, ^[15] rat, ^[16] rabbit ^[17] and human subjects. ^[18] Although MSCs from different species have similar characteristics in part, some data suggest that variations occur among species. MSCs from human bone marrow are relatively easy to harvest and to expand in culture, ^[19] whereas rodent MSCs have proven more difficult, ^{[12],[20],[3]} although this is not without controversy. ^[16] The technical difficulties in preparing MSCs from rodent bone marrow have limited the number of experiments, because animal transplantation models are required for preclinical studies. The selection of suitable cell populations is apparently crucial for the outcome of in vivo experiments with MSCs.

Although there are many methods to isolate MSCs from the bone marrow, no optimal method is available. The methods include plastic adherence, ^[21] density gradient centrifugation ^[22] and immunomagnetic selection. ^[23],[24] Different methods have different defects and virtues. Plastic adherence is an easy method of obtaining such cells on the basis of their plastic adherence characteristics, but it is difficult to get pure stromal cells. Gradient density centrifugation depends on the relative density of MNCs to separate MSCs. Immunomagnetic selection uses the principle of separating the MSC based on the immune recognition of the surface antigens by the use of appropriate antibodies. Extensive experimentation has defined the conditions for the isolation, propagation, and differentiation of MSCs in vitro and in vivo.

In our study we have isolated and established bone marrow stromal cells by the simple and reliable method of combining density gradient centrifugation with plastic adherence and differentiated them to mesenchymal and non-mesenchymal lineages.

Materials and Methods

Animals

Twelve weeks old Wistar rats were used for the experiment. All protocols followed for the use of animals were approved by the Institutional ethical committee and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

Isolation of mononuclear and stromal cells

Rats were sacrificed by cervical dislocation and then placed in 70% alcohol for 10 minutes. Both femurs from one rat were taken and stripped of adherent muscles of the knee end. A needle was inserted into the bone and cells were aspirated followed by several flushes through the bone using a 1 ml syringe filled with culture medium, until all the bone marrow was flushed out of the bone. A similar procedure was performed from the other end of the bone as close to the tip as possible. The marrow thus obtained was suspended by pipetting the large marrow cores through a 1 ml pipette. The cells suspension was divided into two parts one part was used for establishment of culture by plastic adherence while the other half layered over HISTOPAQUE - 1077 (Sigma) and centrifuged on 400g΄30min. Mononuclear cells were removed from the gradient interface and washed with Phosphate buffered saline (PBS). The suspension was then centrifuged at 200g΄5min. The pellet thus obtained was dissolved in 1ml of PBS; the cell count was done in a Neubauer chamber and tested for viability by the Trypan Blue dye exclusion test. The mononuclear cells were resuspended in growth medium (see below), and plated in 25cm ² tissue-culture flasks made of polystyrene plastic (Nunclone) at a density of 1΄10 ⁶ cells/ml. Non-adherent cells were removed after 48 hours, replacing the media every two to three days.

Cell culture conditions

The adherent cells were cultured in the growth medium containing Dulbecco′s modified Eagle′s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; SIGMA) 250KU/L Penicillin, 1.25mg/L Amphotericin-B, 100mg/L Streptomycin, 50μl/L Gentamycin and 1.2g/L Sodium bicarbonate. The cultures were maintained at 37 ⁰ C in a humidified 5% CO ₂ incubator. When the cells reached 80-90% confluency, cultures were harvested with Trypsin-EDTA solution (0.25% trypsin, 1mM EDTA; Sigma).

Colony-forming assays

For these assays, two cells per cm ² at passage 0 were plated and cultured for 14 days in 75 cm ² tissue culture flasks. The cells were fixed with methanol and stained with Giemsa. Colonies less than 2 mm in diameter and those that were only faintly stained were ignored.

Characterization of mononuclear and stromal cells

Immunocytochemistry

MSCs (3 ^rd passage) were seeded into 24 well plates and cultured up to confluency. The cells were fixed with 70% alcohol or 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 20 minutes and then processed for immunocytochemistry. Non-specific reactions were blocked with 5% fetal calf serum for 30 minutes at room temperature. The fixed cells were then incubated for one hour with primary antibodies directed against Vimentin, CD90, CD45, CD11a, CD18, CD34 and CD31. After three washes, cells were incubated with FITC-conjugated secondary antibody for an hour. They were washed three times and counter-stained with propidium iodide (PI) to detect the cell nuclei. Cell were photographed by confocal laser scanning microscopy (LSM510; Carl Zeiss) with a fluorescent light source (excitation wavelength 480 and 540 nm)

Flowcytometry

Mononuclear cells were obtained as described above. MSCs were harvested from the tissue culture flasks after passage 3 in vitro and centrifuged at 200g for five minutes at room temperature. The cells were washed and counted in a Neubauer Chamber. A single cell suspension of 0.5 to 1x 10 ⁶ cells was placed in 50μL of buffer (PBS, 0.1% sodium azide, 2% FBS). The cells were incubated with primary antibody for 40 min with saturating concentrations of monoclonal antibodies CD11a, CD38, CD18, Fibronectin, CD45, Vimentin, and CD90. After the cells were washed three times in buffer and centrifuged at 200 g for five minutes, they were resuspended in ice cold PBS and incubated with the FITC-labeled or TRITC-labeled secondary antibody for 30 minutes in the dark at 4 ⁰ C. Cell fluorescence was evaluated by flow cytometry in an FACS Calibur instrument (Becton Dickinson) and the data were analyzed using Cell Quest software (Becton Dickinson). An isotype control was included in each experiment and specific staining was measured from the cross point of the isotype with a specific antibody graph.

RT-PCR

Total RNA was extracted from the 3 ^rd passage of rat marrow stromal cultures using Trizol ^TM according to the manufacturer′s protocol. The RNA so obtained was evaluated by spectrophotometry and the integrity was checked by gel electrophoresis. Total RNA (1 μg) was reverse transcribed using Murine Leukemia Virus Reverse Transcriptase (Fermantas, #EPO451) for 30 minutes at 42°C in the presence of oligo-dT primer. The PCR was carried out using 2μl of cDNA in a total volume of 25μl. The final concentrations of the PCR components were dNTPs, MgCl ₂ , each sense and antisense primer and Taq DNA polymerase. PCR was performed with primers [Table - 1] for 30-35 cycles, each cycle consisting of denaturing at 95°C for 30 seconds, annealing at 55-60°C for 30 seconds, and elongation at 72°C for 1 minute, with an additional 10-minute incubation at 72°C after completion of the last cycle. To exclude possible contamination of genomic DNA, PCR was also applied to reactions without RT. The amplified complementary DNA was electrophoresed through a 1.5% agarose gel, stained and photographed under ultraviolet light.

Differentiation

Adipogenic differentiation

Passage 2 cells were seeded on cover slips in six well plates and cultured in complete medium up to confluency. At confluency, the cells were switched to an adipogenic medium (DMEM/10% FBS supplemented with 0.5 μM dexamethasone (Sigma, USA) 0.5 mM isobutylmethylxanthine (Sigma, USA), and 10 μg/ml insulin (Sigma, USA)) and further cultured upto 21 days with the medium being changed on every alternate day. After 21 days, the adipogenic cultures were fixed in 4% paraformaldehyde for atleast one hour and stained with fresh 0.3% oil Red-O solution for two hours. After staining, the cultures were washed three times and counter stained with hematoxylin.

Osteogenic differentiation

Passage 2 cells were seeded on cover slips in six well plates and cultured in complete medium up to confluency. The medium was then replaced with a calcification medium containing DMEM/10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 μm ascorbic acid (Sigma, USA) and incubated for 21 days. These cover slips were stained with fresh 0.5% alizarin red solution.

Neural differentiation

Passage 2 cells were used for neural differentiation. At confluency, the cells were switched to DMEM+ITS for 24 hours. After 24 hours, the neurogenic induction medium (DMEM+ITS+EGF+bFGF+NGF) was added. After six to seven days the cultures were terminated and processed for immunocytochemistry.

Results

Isolation and culturing of MSCs

By plastic adherence: The cells suspension containing both stromal and hematopoietic cells was seeded in tissue culture flasks using DMEM with 10% FCS. At the end of two days, many of the rounded as well as spindle shaped cells had attached to the base of the tissue culture flask. Even on subsequent media change most of these rounded cells remained adherent [Figure - 1]a.

Ficoll hypaque separation and plastic adherence (combination method): The cell suspensions were layered on hypaque and subsequently seeded in tissue culture flask. The spindle shaped cells attached to the bottom of the flask while the round cells remain suspended in the medium and were mostly eliminated from the culture with subsequent media changes [Figure - 1]b. Majority of the adherent cells displayed a spindle like shape [Figure - 1]c. These cells began to proliferate at about day 4 and gradually grew to form small colonies [Figure - 1]d. By day 7, the number of cellular colonies of different sizes had obviously increased. In large colonies, cells were more densely distributed and showed a spindle shape [Figure - 1]e. As cells continued to grow, colonies gradually expanded in size and reached confluency by day 10. Passaged MSC behavior was similar to that in primary culture. However, the cells were larger and more heterogeneous in morphology and growth properties. Grossly, the MSCs in subcultures could be divided into two types, spindle shaped and broad flattened cells [Figure - 1]f. The flattened cells seldom proliferated and were gradually surrounded by the spindle shaped cells, which replicated faster. It seemed that the spindle-like MSCs gradually transformed into broad flattened cells with further passages. When seeding at low density, the cells form colonies [Figure - 1]g-h. The colony forming efficiency was counted as 10%.

Characterization

Flow Cytometry

The mononuclear cells expressed CD45, CD11a, CD18, CD31 [Figure - 2] and suggest hematopoietic lineage. The adherent marrow stromal cells expressed Vimentin, Fibronectin and CD90 [Figure - 3]. They expressed neither hematopoietic lineage markers such as CD45, CD11a, CD18 nor an endothelial related antigen CD31 [Figure - 3]. The lack of expression of CD45, CD11a, CD18 and CD31 suggests that cell cultures were depleted of hematopoietic cells during sub-cultivation. [Table - 2] summarizes the expression of markers by stromal cells isolated by solo density gradient centrifugation, solo plastic adherence and combination of both.

Immunocytochemistry

The mononuclear cells showed a high nucleus to cytoplasmic ratio [Figure - 3]a. Immunochemistry examination clearly detected the localization of CD34, CD45, CD11a, CD18 and CD31 on bone marrow mononuclear cells and vimentin and CD90 on marrow stromal cells [Figure - 3]. MSC′s were negative for the markers CD45, CD11a, CD18 and CD31 [Figure - 3].

RT-PCR

RT-PCR results showed expression of vimentin and collagen type alpha 1 in isolated BMSCs [Figure - 4]. This shows that the isolated cells are genuine marrow stromal cells with little or no contamination from other bone marrow cells such as hematopoietic cells.

Differentiation

Adipocytic, osteocytic differentiation

BMSCs were differentiated in vitro using adipogenic and oesteogenic induction media. Following three weeks of adipogenic induction, the cells stained oil red ′O′ positive showing lipid laden adipocyte phenotype [Figure - 5]. Similarly, when induced with oesteogenic induction medium for two to three weeks, these cells showed oesteogensis upon staining with alizarin red for calcium deposits [Figure - 6].

Neural differentiation

MSCs when induced with neural differentiation media for eight days under serum-free conditions started showing neuron like morphology by day 4 with slender dendritic processes and characteristic aura around soma. The differentiated neural cells stained positive for neural markers like Nestin, Tuj1/Beta tubulin-III, Neurofilament, Synaptophysin on immunocytochemistry [Figure - 7].

Discussion

There are many methods to isolate stromal cells from bone marrow, including plastic adherence, ^[21] gradient density centrifugation ^[22] and Immunomagnetic selection. ^[23],[24] Different methods have their own limitations and advantages. For example, plastic adherence is an easy method of obtaining such cells on the basis of their plastic adherence characteristics, but it is difficult to get pure stromal cells. As shown in [Figure - 1]a, we observed adherence of other cells along with spindle shaped cells. Gradient density centrifugation depends on the relative density of MNCs to separate MSCs. Immunomagnetic selection uses MSC receptors and antigens. Other methods have also been used to isolate MSCs, ^[25],[26] but none of these were found to be optimal. In this study we used the Ficoll (1.077g/ml) method to isolate MSCs from bone marrow aspirate. After centrifugation, we found many suspended cells in the medium for 72 hrs. This could be due to the density of cells, which was changed slightly in DMEM. Therefore we combined the density gradient centrifugation with plastic adherence and changed the medium three times to obtain a purer isolate of MSCs after the density gradient centrifugation. According to the results, the combination method is relatively simple and can easily be used to obtain pure MSCs.

MSCs were first described in 1968 by Friedenstein et al.^[27] who discovered that MSCs adhered to tissue culture plates, resembled fibroblast in their morphology and formed colonies. ^[12] These characteristics have been identified in MSCs from numerous species including human, rat, mouse, rabbit and monkey. However, the expandability of MSCs in vitro varied dramatically among different species and different methodologies for isolation and plating of the cells. In our study, the MSCs adhered to the plate and had a fibroblast spindle-shaped morphology, forming colonies when grown in the low plating density. A small number of MSCs have a broad flattened shape.

Several groups have illustrated the multipotentiality of rat bone marrow MSCs and their usefulness as sources for cell therapy. For example, Woodbury et al.^[28] stimulated rat MSCs to differentiate into neurons by plating rat MSCs at 8,000 cells/cm ² and growing them to confluency. Passage 6 cells were then used for neuron differentiation. ^[28] Hofstetter et al.^[29] implanted rat MSCs into the spinal cord. They plated rat MSCs at 5,000cells/cm ² and grew them to conflluency. Passage 5 cells were used for implantation. ^[29] Dezawa et al.^[30] induced rat MSCs to differentiate into Schwann cells in vitro and implanted into the sciatic nerve. Rat MSCs were subcultured four times and used. ^[30] None of the authors noted the quantum of increase of rat bone marrow MSCs; however, the cells had to be replated more than four times to harvest enough cells for their purposes. One report ^[31] obtained 10 ⁸ cells at passage 4 with the initial density of 6000 cells at passage 2. In our study, we started with 10,000 cells in 75 cm ² flasks at passage 1 and obtained 10 ⁸ cells at passage 3. These cells seem to be sufficient for in vitro or transplantation analysis. These data indicate that the proliferation ability of our rat bone marrow MSCs compares favorably with those in previous reports. However, it needs to be seen whether these cells consist of a single or mixed population of stromal cells.

The isolated stromal cells were positive for CD90, Vimentin, fibronectin and negative for CD45, CD11a, CD31 and CD18, which are the markers of hematopoietic lineage as previous reports. ^[22-24] On comparing the level of expression of mesenchymal markers in cells obtained by the three methods we have observed a higher percentage of expression in cells isolated using combination method. In case of cultures established using plastic adherence property, the number of CD90 positive cells were observed to be 24.4% and those positive for hematopoietic markers CD11a was 3.7%, CD45 (15.4%) and CD18 (30.9%). However, in case of cells isolated using a combination method, the expression of CD90 was 84% and hematopoeitic markers CD11a was 1.5%, CD45 (6.7%), CD31 (0.4%) and CD18 (2.7%). This implies that the combined method gives pure stromal cells in comparison to plastic adherence. Prior gene expression profile studies of BMSCs, including microarray analysis, have shown that certain genes such as vimentin and collagen type 1 alpha 1 are selectively enriched in these cells. ^{[25],[26],[27]} Therefore we selected these genes that confirm the identity of isolated cells. As the RT-PCR results show, these transcripts are expressed in isolated BMSCs. This indicates that the isolated cells are genuine, non-hematopoietic marrow stromal cells. Based on the phenotypic analysis by IF and FACS and RT-PCR analysis, particularly the presence of CD90, fibronectin and Vimentin antibodies, there is a strong suggestion that these mesenchymal cells have stem cell like characteristics. The self-renewal capacity up to an average of 20 passages also points towards the increased potential for proliferation and some of the cells so obtained may prove to possess stem cell properties.

The isolated stromal cells showed differentiation into other mesenchymal lineages adipogenic and osteogenic as observed earlier groups. ^{[11],[13],[32],[33],[34],[35],[36]} We have also tried to differentiate them into neuronal like cells. These cells form neuronal like process and ultimately express synaptophysin during neuronal differentiation, suggesting acquisition of a mature neuronal phenotype upon induction of differentiation. In addition, the expression of mature neural marker neurofilament confirms the capacity of BMSCs to differentiate into neuronal like cells.

Our observations indicate that rat MSCs retain the capacity to differentiate into non-mesenchymal derivatives, specifically neurons, suggesting that intrinsic genomic mechanisms of commitment, lineage restriction, and cell fate are mutatable. Environmental signals apparently can elicit the expression of pluripotentiality that extends well beyond the accepted fate restrictions of cell originating in classical embryonic germ layers. These adult cells are self-renewing and multipotential, ^{[11],[13],[32],[33],[34],[35],[36]} thereby fulfilling many of the criteria of a stem cell population. Mesenchymal cells can differentiate into neurons in vitro. MSCs may be useful in the treatment of a wide variety of neurologic diseases, offering significant advantages over other Stem cells. MSCs grow rapidly in culture, precluding the need for immortalization and differentiate into neurons exclusively with use of simple protocol.

In summary, by the simple principle of adhesion, it is possible to establish an efficient method of harvesting a fairly homogenous population of bone marrow stromal cells; the phenotypic characteristics and differential potential of these cells point towards the stem cell like features.

Acknowledgments

CSIR, New Delhi, India is acknowledged for providing a fellowship to the first author. The authors thank the Department of Biotechnology (DBT) for financial support and the Hyderabad Eye Research Foundation for extending the financial support.

References

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