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Isolation, expansion, and characterization of mesenchymal stem cells from adult rat bone marrow

Hammed, Samy Hosnya; El Shawarby, Amany Mohameda; Elrahman Ahmed, Mohamed Abda; Abo Golayel, Mohamed Kamelb; Elmonem Mohamed, Asmaa Abda

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The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 281-290
doi: 10.1097/01.EHX.0000396875.59556.15
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Abstract

Introduction

Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that are present in the bone marrow. They are few populations representing 1 in 10 000 of nucleated cells. They have the ability to expand many folds in culture while retaining their growth and multilineage potential [1].

The presence of nonhematopoietic stem cells in the bone marrow (BM-MSCs) was first observed by Cohnheim 130 years ago. He suggested that BM may be the source of fibroblasts that deposit collagen fibers as part of the normal process of wound repair. Non-hematopoietic progenitor cells were found in the BM called colony forming unit fibroblasts. These cells were a heterogeneous population of progenitors with plastic adherence properties that can differentiate both in vitro and in vivo to osteogenic, chondrogenic, and hematopoietic stromal supportive cells [2]. However, similar populations have been reported in other tissues, such as cord blood, trabecular bone, adipose tissue, dental pulp, muscle, and brain [3].

MSCs express a number of specific surface markers such as CD29, CD44, CD71, CD105, CD106, and CD166. It is generally agreed that adult human MSCs do not express the hematopoietic markers CD45, CD34, CD14, or CD11 [4].

MSCs have gained considerable attention due to their potential use for cell replacement therapy and tissue engineering [5]. Cultured MSCs have been administered systemically to humans to treat osteogenesis imperfecta, Parkinson's disease, and inflammatory bowel disease [6–8]. Moreover, murine BM-MSCs could serve as vehicles for interleukin-12 gene deliveries in Ewing sarcoma treatment [9]. Hence, MSCs are easily isolated from adult tissues that are not ethically restricted and have low immunogenicity [10]. In addition, there was no evidence of malignant transformation in any reimplant site in patients treated for various orthopedic conditions with culture expanded BM-MSCs [11].

This trial aimed to isolate, increase the expansion rate, and characterize BM-MSCs immunohistochemically and by electron microscopy.

Materials and methods

Animals

Twenty adult male albino rats weighing 150–200 gm were used in this study. The animals were kept under good illumination and ventilation conditions in the animal house. The rats were killed according to the Ethics Committee recommendations of Ain Shams University. All the experiments were conducted in the Medical Research Center, Ain Shams University Hospitals.

Chemicals

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin solution, and 0.25% trypsin–0.02% EDTA solution were purchased from Sigma-Aldrich Co., St Louis, USA. Phosphate-buffered saline (PBS) was purchased from Lonza Bioproduct, Belgium. Ultra V block, mouse monoclonal antibodies for CD44, CD105, CD34, and biotinylated goat antimouse secondary antibody were purchased from Thermo Scientific, Fremont, USA (all other general reagents used and all dishes and flasks were of tissue culture quality).

Separation of the rat serum from the peripheral blood

The rats were anesthetized by diethyl ether inhalation and their necks were sprayed with 70% ethanol. The hairs of the neck were shaved using a sterile scalpel. Cleaning of the skin of the neck was performed using 70% ethanol and opened by a sterile scalpel to show the neck vessels. The neck vessels were punctured using a sterile syringe to obtain 3–4 ml of blood. The blood samples were then transferred immediately into a sterile tube. The blood samples were centrifuged at 8000 rpm at room temperature for 10 min to separate the serum [12]. The serum was aspirated carefully using a sterile pipette and was added to the complete medium.

Harvesting of the rat bone marrow, isolation, and culture of mesenchymal stem cells

Isolation and primary culture of MSCs from the long bone of donor rats were performed using Caplan's method [13]. The isolation process was carried out in a laminar flow cabinet under strict sterile conditions. The femoral and tibial bones were harvested from the 20 rats and divided equally into two groups:

  • (1) Group 1: both ends of the long bones were cut away from the diaphyses. The BM was flushed out from the bone with complete medium, consisting of DMEM containing 10% FBS, penicillin G (100 U/ml), and streptomycin (100 μg/ml).
  • (2) Group 2: the BM was flushed out from the bone with complete medium containing 8% FBS, penicillin G (100 μg/ml), streptomycin (100 μg/ml), and 2% autologous rat serum.

The marrow plugs of both groups were then dissociated by pipetting and the dispersed cells were centrifuged at 1800 rpm for 10 min and the cell pellets were resuspended in 2 ml of complete medium. A sample of 100 μl of the cell suspension was dispensed on top of a hemocytometer slide so that the fluid entirely covered the surface of the squares of the slide. The number of cells was then counted, using the ordinary light microscope, in the nine squares. The total cell number was calculated according to the following equation [14]:

These cells were then seeded into 35 mm petri dishes at a density of 5×105 cells/cm2 and incubated at 37°C in a humidified incubator containing 5% CO2. The cultured cells were examined daily under the phase contrast microscope to detect the appearance of any infection. Seven days after culture, the nonadherent cells and the medium were discarded by aspiration. The adherent cells were washed with DMEM and 2 ml of fresh complete medium was added to the dish. Culture medium was replaced every 3 days. When primary cultures became nearly confluent (80–90%), the cells were detached by adding 0.5 ml of 0.25% trypsin containing 0.02% EDTA for 5 min. The action of the trypsin EDTA was then blocked by adding 2 ml of complete medium and the cell suspension was subcultured into three new 35 mm petridishes at a density of 1×105 cells/cm2. On day 12 from the primary culture, aliquots of the cells were prepared to be examined by Giemsa stain, transmission electron microscopy, and immunohistochemical staining for CD44, CD105, and CD34.

Staining of mesenchymal stem cells by Giemsa stain

On day 12 postseeding, the medium within the 35 mm petridish was aspirated and the adherent MSCs was washed twice with PBS. The cells were fixed by adding freshly prepared precooled (−20°C) mixture of acetone (at a ratio of 1 : 1) for 15 min in the freezer [15]. The mixture was aspirated and the fixed cells were washed twice with PBS and stained by adding 2 ml Giemsa stain for 1 h and then washed with tap water. Photographs were taken by using the phase contrast microscope (Axiovert 100, Carl-ZEISS, Jena, Germany).

Transmission electron microscopy

Ultrastructural detection of the cultured MSCs was carried out on day 12 postseeding. The medium was aspirated and the adherent MSCs was washed twice with PBS. The cells were detached by trypsinization, washed with complete medium, and centrifuged at 1800 rpm for 5 min. The cell pellet was resuspended and fixed with 2.5% glutaraldehyde in 0.1 mol/l PBS (pH 7.4) for 2 h at room temperature. After centrifugation, the pellet was then washed with PBS and postfixed in 1% osmium tetroxide, dehydrated in ascending grades of alcohols, and embedded in epoxy resin. Ultrathin sections were cut at 60 nm and stained with 2% uranyl acetate for 10 min followed by lead citrate for 10 min [16]. Images were captured with a Jeol, JEM-1200 EX II Electron Microscope, Tokyo, Japan.

Immunohistochemistry staining

The cultured MSCs at day 12 from primary culture were fixed with a precooled mixture of acetonefor 15 min in the freezer. Nonspecific background staining was avoided by incubation of the cells with Ultra V block for 5 min. Then, the cells were incubated for 2 h with the primary antibodies for CD44, CD105, and CD34. After washing with PBS, cells were incubated with biotinylated goat antimouse immunoglobulin G (1 : 200) secondary antibody. The site of antibody immunostaining was detected by adding streptavidin peroxidase for 30 min. Freshly prepared diaminobenzidine was used as a chromogen to visualize the antibody binding [17]. The positive cells showed brownish discoloration of the cytoplasm and the cell processes. Images were captured by the phase contrast microscope.

Results

Morphological identification of bone marrow-mesenchymal stem cells using phase contrast microscopy

Twenty-four hours from the primary culture (passage 0=P0) of rat BM-MSCs, the cultured cells in groups 1 and 2 appeared crowded and suspended. They were variable in size and shape. Most of the cells appeared rounded.

Three days from the primary culture, MSCs of group 1 were seen attached to the culture dishes sparsely and sporadically. The cells appeared spindle shaped (Fig. 1). In contrast, MSCs of group 2 were arranged in the form of small colonies. They were of variable shapes (spindle, star, and triangular) with many processes, granular cytoplasm, and rounded vesicular nuclei (Fig. 2).

Figure 1
Figure 1:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cellss on day 3 from the primary culture showing few cells starting to attach sporadically. Some cells appear spindle shaped (▴), whereas others start to form processes (↑). Observe that the nonadherent cells appear rounded and retractile (↑↑). Group 1 (P0 ×200).
Figure 2
Figure 2:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells on day 3 from the primary culture, showing the cultured cells appear to form small colonies. They are spindle shaped (▴), others appear star shaped (*) and triangular in shape with many processes, granular cytoplasm, and rounded vesicular nuclei (↑). Observe that the nonadherent cells appear rounded and retractile (↑↑). Group 2 (P0 ×200).

Seven days from the primary culture, the cells of group 1 proliferated and reached 40% confluency. The cells became plump with well-developed cytoplasmic processes, granular cytoplasm, and vesicular nuclei (Fig. 3). The cultured cells of group 2 reached nearly 70% confluency and expressed different shapes. Some cells appeared triangular in shape with long multiple processes. Other cells appeared spindle shaped and star shaped. Most of these cells showed granular cytoplasm and central vesicular nuclei (Fig. 4).

Figure 3
Figure 3:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells on day 7 from the primary culture showing that the cultured cells begin to form small colonies and reach 40% confluency. Some cells are spindle shaped (▴), others appear star shaped (*) and triangular in shape with many processes, granular cytoplasm, and rounded vesicular nuclei (↑). Observe that the nonadherent cells appear rounded and retractile (↑↑). Group 1 (P0 ×200).
Figure 4
Figure 4:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells on day 7 from the primary culture showing the cells reach approximately 70% confluency. Some cells appear spindle shaped (▴), others appear star shaped (*) and triangular in shape (↑) with granular cytoplasm and rounded vesicular nuclei. Group 2 (P0 ×200).

Twelve days from the primary culture, the adherent cells of group 1 reached 60–70% confluency. As growth of the cells continued, colonies gradually expanded and interconnected to each other. The cells were spindle, triangular, and star shaped (Fig. 5). In contrast, the adherent cells of group 2 reached 90% confluency and appeared triangular, star, and spindle shaped (Fig. 6).

Figure 5
Figure 5:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells 12 days postseeding showing the cultured cells reach approximately 70% confluency. Most of the cells appear spindle shaped (▴), others appear star shaped (*) and triangular in shape (↑). Group 1 (P0 ×200).
Figure 6
Figure 6:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells 12 days postseeding showing the cells are approximately 90% confluent. Most of the cells appear spindle shaped (▴), others appear star shaped (*) and triangular in shape (↑) with granular cytoplasm and rounded vesicular nuclei. Observe the binucleated dividable cells (↑↑). Group 2 (P0 ×200).

The first passaged MSCs (P1) of group 2 were obviously larger in size and more or less homogenous in morphology and appeared as small spindle, triangular, and as broad flattened cells (Fig. 7).

Figure 7
Figure 7:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells 14 days after passage 1 (P1). The cells are homogenous in appearance. Most of them appear spindle shaped (▴) and triangular shaped (↑). Observe the appearance of few broad flattened cells (↑↑). Group 2 (P1 ×200).

Twelve days from the primary culture, the cells were stained with Giemsa stain. The adherent MSCs appeared star shaped with multiple interdigitating processes, bluish granular cytoplasm, and their nuclei were rounded with prominent nucleoli. Some of these cells were binucleated indicating evidence of cellular division (Fig. 8).

Figure 8
Figure 8:
Phase contrast micrograph of a primary culture of rat bone marrow-mesenchymal stem cells 12 days after isolation and culture showing star-shaped and triangular cells interconnected together with cytoplasmic processes. The cells show bluish granular cytoplasm and bluish vesicular nuclei. Observe the binucleated cells (↑). Group 2, Giemsa stain (P0 ×200).

Immunohistochemical identification of bone marrow-mesenchymal stem cells

Twelve days postseeding, the MSCs appeared branched with a positive brownish cytoplasmic immune reaction for CD44 (Fig. 9). On using primary antibody for CD105, the cells also showed positive brownish reactivity (Fig. 10). In contrast, these cells did not show any immune reaction for the surface marker, CD34, specific for the hematopoietic stem cells (Fig. 11).

Figure 9
Figure 9:
Phase contrast micrograph of primary culture of rat bone marrow-mesenchymal stem cells 12 days after isolation and culture showing branched cells with positive cytoplasmic immune reactions for CD44. Group 2, avidin–biotin peroxidase for CD44 (P0 ×200).
Figure 10
Figure 10:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells 12 days after primary culture showing that most of the cultured cells appear with positive brownish cytoplasmic immune reaction for CD105. Group 2, avidin–biotin peroxidase for CD105 (P0 ×200).
Figure 11
Figure 11:
Phase contrast micrograph of rat bone marrow-mesenchymal stem cells 12 days after primary culture showing that all of the cultured cells are negatively immunostained for CD34. Group 2, avidin–biotin peroxidase for CD34 (P0 ×200).

Ultrastructural examination of bone marrow-mesenchymal stem cells

Twelve days from the primary culture, the native MSC showed irregular euchromatic nucleus. The cytoplasm was rich in free ribosomes, rounded mitochondria, prominent Golgi apparatus, and few rough endoplasmic reticulum (rER) (Fig. 12). Some cells appeared rounded with an irregularly shaped plasma membrane forming multiple pseudopodia. The nucleus was small and eccentric with peripheral heterochromatin and central euchromatin. The cytoplasm was rich in rounded and elongated mitochondria, numerous free ribosomes, and lysosomes (Fig. 13). Some cells appeared with two euchromatic elliptical nuclei, which are situated peripherally. The cytoplasm was rich in free ribosomes, some rER, and elongated mitochondria (Fig. 14). Other cells showed few pseudopodia arising from the plasma membrane. The nucleus was rounded and central with thin peripheral heterochromatin and large central euchromatin. The cytoplasm contained rounded mitochondria, lysosomes, and some vacuoles (Fig. 15).

Figure 12
Figure 12:
An electron micrograph of rat bone marrow-mesenchymal stem cell 12 days postseeding showing that the cytoplasm is rich in free ribosomes, rounded mitochondria (M), few rough endoplasmic reticulum (↑), and supranuclear Golgi complex (G). The nucleus is irregular and euchromatic (N). Group 2, TEM (×12000).
Figure 13
Figure 13:
An electron micrograph of rat bone marrow-mesenchymal stem cell 12 days after primary culture showing rounded cells with an irregular shaped plasma membrane forming multiple pseudopodia (↑). The cytoplasm is rich in rounded mitochondria (M), numerous free ribosomes, and lysosomes (L). The nucleus is small and eccentric with a central area of euchromatin and peripheral heterochromatin (N). Group 2, TEM (×7500).
Figure 14
Figure 14:
An electron micrograph of rat bone marrow-mesenchymal stem cell 12 days postseeding showing a rounded cell with irregular cell membrane. The cytoplasm is rich in rounded mitochondria (M), some rough endoplasmic reticulum (↑), numerous free ribosomes, and few lysosomes (L). There are two elliptical euchromatic nuclei, which are situated peripherally indicating active cell division (N). Observe the large nucleolus (↑↑). Group II, TEM (×7500).
Figure 15
Figure 15:
An electron micrograph of rat bone marrow-mesenchymal stem cell 12 days postseeding showing rounded cell with few pseudopodia (↑). The cytoplasm is rich in rounded and elongated mitochondria (M), lysosomes (L), and some vacuoles (▴). The nucleus is rounded and central with thin peripheral heterochromatin and large central euchromatin. Group 2, TEM (×7500).

Discussion

Classically, the optimal conditions for MSC expansion require media supplemented with 10% FBS [18]. Many investigators used DMEM, supplemented with 10% FBS and penicillin/streptomycin as a culture medium for growth of BM-MSCs [19,20]. Although some investigators used α-modified minimal essential medium supplemented with 20% FBS, others used Iscove's modified Dulbecco's medium supplemented with 20% FBS [21].

Most cultured cells need serum to grow. In our trial, to increase the yield and expansion rate of isolated rat MSCs in culture, we supplemented DMEM with 2% autologous rat serum in group 2. The proliferation of MSCs started earlier after 3 days from the primary culture and increased progressively by day 7. The cells reached 90% confluency on day 12. In contrast, the cells of group 1 started to proliferate later than those of group 2 and failed to reach 90% confluency by day 12. Increase of the expansion rate of cultured MSCs in this study is due to the autologous rat serum we added. It contains several growth factors that are responsible for growth-promoting effects, such as somatomedins, multiplication-stimulating activity, and insulin-like growth factors (IGFs). These growth factors lead to a rise of DNA, RNA, protein synthesis, and finally to cell replication. All of these polypeptide hormones are members of one family and they do not exist in the serum in free form, but are bound to specific carrier proteins. These proteins are present in great excess in the serum that probably serves as a storage pool [22]. Moreover, there is an important biological property of the IGF-binding proteins, which is their ability to increase the circulating half-life of the IGFs that act as potentiators of cell proliferation [23].

Cell plating density is a critical parameter to ensure good expansion rate of MSCs [24]. In this study, the cells were seeded into 35 mm tissue culture petridishes at a relatively high density of 5×105 cells/cm2. This was explained by some investigators who stated that a much lower initial seeding density might delay the establishment of autocrine and paracrine growth factors secreted by MSCs in culture [25].

In this study, 3 days postseeding, the MSCs of group 1 began to adhere to the plastic surface singly and sporadically and most of them were spindle shaped. In contrast, the cultured cells of group 2 (received rat serum) started to form small colonies that began to interconnect to each other. Our finding is in agreement with a previous study that detected the initial adherent spindle-shaped cells appearing as individual cells or clusters of a few cells on day 3 [21].

In this study, 7 days after primary culture, the adherent rat BM-MSCs of group 1 began to proliferate forming small colonies and became 40% confluent. In contrast, BM-MSCs of group 2 proliferated more rapidly and reached approximately 70% confluency. This came in accordance with a previous study reporting that the MSCs began to form colonies 7–10 days after initial plating [26].

The culture medium, in this study, was firstly changed after 7 days from the primary culture to allow adherence and proliferation of the MSCs, unlike some investigators who changed the medium after 3 days [27]. Washing of the cultured MSCs was performed by using sterile PBS and replacement of the culture medium was performed every 3 days. The same procedures were carried out by many investigators [28,29].

Twelve days after initial plating, the adherent MSCs of group 1 in our study reached only 60–70% confluency and showed spindle, triangular, and star shapes. MSCs of group 2 continued to grow forming large colonies that were gradually expanded and interconnected to each other and reached 90–95% confluency. In contrast, a previous study reported that the cultured MSCs expanded in size and reached 95% confluency at 21–28 days after initial plating, depending on the proliferating ability of each sample [17].

In this study, after 12 days from the primary culture, the MSCs were stained by Giemsa stain. The adherent cultured cells appeared star shaped with bluish granular cytoplasm, multiple interdigitating processes, and their nuclei were rounded with prominent nucleoli. Some of these cells were binucleated indicating evidence of cellular mitotic division. The bluish granular cytoplasm might be due to free abundant ribosomes. Similar findings were described by previous studies [19,26].

The adherent cells of group 2 in this study were subcultured when they reached 90–95% confluency. The first passaged MSCs (P1) were larger in size and homogenous in morphology. The cells in the subculture were small spindle, triangular, and broad flattened cells. Our results came in agreement with the results of some investigators who detected that the passaged MSCs behaved similar to those in primary cultures [25]. They divided the MSCs in the subculture into two types, small spindle or trianglular in shape and broad flattened cells. They added that the broad flattened cells proliferated rarely. Appearance of the broad flattened cells was explained by gradual transformation of the spindle and triangle-like MSCs into broad flattened cells with passages [25].

MSCs were identified by the expression of many molecules and surface markers that are specific for them. In this study, characterization of rat BM-MSCs was detected by immunohistochemical staining using CD44 and CD105 antibodies on day 12 from the initial plating. MSCs appeared branched with positive brownish cytoplasmic immune reaction. In contrast, MSCs did not show any reaction for the surface markers specific for hematopoietic stem cells such as CD34 antigen. This suggested that there were no hematopoietic cells in the culture. Similar results were detected by some researchers [27].

MSCs on day 12 revealed multiple small pseudopodia arising from the plasma membrane. Some researchers stated that these pseudopodia provided capacity for the cells to migrate within the receiving tissue [30,31].

In this study, some cells showed rounded and eccentric nuclei with thin peripheral chromatin at the inner nuclear membrane and large central euchromatin. The inner part of the cytoplasm was rich in free ribosomes, some lysosomes, few rER, obvious Golgi complex, and rounded mitochondria. Other cells appeared rounded with central nucleus and few pseudopodia. This came in agreement with some researchers who mentioned that there were two ultrastructural features distinguishing MSCs from fibroblasts: the eccentric, irregularly shaped nucleus and the relative richness of the inner cytoplasmic zone with mitochondria and Golgi apparatus [32]. In general, the ultrastructural appearance of the MSCs indicates that they are stem cells in a relatively advanced state of differentiation.

In this study, some cells appeared with two eccentric, euchromatic and elliptical nuclei. Some investigators reported that MSCs were frequently binucleated [33,34]. However, investigators suggested that this observation might be attributed to the irregular shape of the nucleus and not due to the presence of two nuclei [32]. A later study reported that some binucleated MSCs indeed existed in the culture [35].

In this study, cytoplasmic vacuoles in some MSCs were observed. This came in accordance with previous studies that explained formation of the vacuoles due to dilatation in the endoplasmic reticulum and Golgi apparatus [32,35].

In conclusion, our results indicated that MSCs derived from adult rat BM can be easily isolated and expanded in culture more rapidly using 2% rat serum. Hence, we recommend supplementing the culture media with rat serum.

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Keywords:

CD105; CD44; mesenchymal stem cells; rat bone marrow; rat serum

© 2011 The Egyptian Journal of Histology