INTRODUCTION
In regenerative medicine, stem cells are considered a very promising therapeutic agent; thus, they intensely studied (1). Translational research with stem cells seeks to find therapeutic alternatives that contribute to the solution of tissue and organ loss (2). In many cases, the study of these alternatives includes the use of animal models (3).
Stem cells exist in different pre- and postnatal tissues, both in humans and in animals. Unlike other cell types, such as somatic cells, stem cells possess a self-renewal capacity, non-differentiated status, and potential to differentiate into almost any type of cell (4,5). Additionally, great therapeutic potential in regenerative medicine has been attributed to stem cells due to their secretion of cytokines and growth factors (6). To guarantee the safe use of these cells in clinical practice, the scientific community is interested in deepening the knowledge of stem cell origin, location, isolation methods, in vitro and in vivo characterization, applications, and mechanisms of therapeutic action (7).
Stem cells have received different names. The most used term mesenchymal stem cells (MSCs) (8-10), which was first introduced in 1991 by Arnold Caplan (11). Currently, because its main functions relate to its immunomodulatory properties, scientific societies have suggested changing the meaning of MSC (12,13) to medicinal signaling cells (14) while retaining the acronym MSC through which they have been widely known.
MSCs were first described in bone marrow, which is why this tissue has been the main source for obtaining them and has been one of the most intensively studied (11,15,16). Currently, MSCs can be recovered and isolated from various organs and tissues (12,14), such as placenta (17), umbilical cord blood (18,19), umbilical cord (20), adipose tissue (21), skin (22), testicles (23), brain (24), and dental pulp of permanent and deciduous teeth (25-27). Even though common characteristics between MSC populations have been reported from various sources, there is not yet an updated consensus of specific criteria to define them (28, 29). Stem cells derived from adult tissues have the ability of immunomodulating and establish cells without the risk of generating teratomas, in contrast to embryonic stem cells or induced pluripotent stem cells.
MSCs from bone marrow have been widely investigated and used in cell-based therapies (12, 30). The U. S. Food and Drug Administration has approved their use in autologous applications for the treatment of various diseases (31). For the application of MSCs in regenerative medicine, a rigorous process of obtaining, isolating, and characterizing stem cells seeks to guarantee their conservation (8), in addition to maintaining established MSC cultures from a primary culture to their successive passages. This is still a complex process (29) due to the presence of other cell types such as fibroblasts, which are morphologically identical and present surface markers like those of MSCs (32). The characterization of stem cells must be completed with the determination of their multi-differentiation potential (8,33,34) and, to guarantee their translation, studies seek to determine their efficacy and safety in different therapeutic applications (29).
Because in vitro models do not fully mimic the complexity of an in vivo environment or predict the clinical efficacy of stem cell treatments, many studies use animal models (35). Animal models are necessary to assess innovative ideas, concepts, and technologies. Regenerative medicine, for example, has advanced because of experiments conducted with animal models (35). Rats are a model organism to study physiological functions in vivo; in both pathological and disease conditions the rat model is more comparable to humans than mice (36). Rats were the first species among mammals to be domesticated for scientific research with work dating back to before 1850 (37). The first homozygous inbred strain obtained from brother-sister mating was established in rats by King in 1909. Inbred work in mice began that same year (37).
Recent advances in genetic engineering have resulted in the development of various immunodeficient rat models to transplant and regenerate human tissues and cells (38,39). Due to their size and tissue density, these models caused the advancement of techniques such as multiphoton microscopy (36) and electrocardiography (40). Currently, there are models of immunocompromised rats transplanted with human immune cells that are known as humanized rats, which provide a means for robust longitudinal studies on the safety and efficacy of therapeutic agents aimed at the treatment of HIV and modeling of cardiovascular, neurocognitive, and pulmonary diseases (41).
Considering that rat is a species that allows applying preclinical models close to humans (42), it has been the selected model for our research group to develop in vitro and preclinical methods, which require identification, characterization, and proper manipulation of bone marrow-derived stem cells. Therefore, the research question that motivated this study was: What are the procedures to maintain rat’s bone marrow cell cultures that maintain an immunophenotypic predominance of stemness, differentiation potential to different lineages, and allows obtaining it in high cell densities to be used in regeneration experiments? With the completion of this study, we developed laboratory methods to isolate, cultivate, expand, and functionally characterize bone marrow-derived stem cells, which contribute to develop dental tissue-regeneration protocols in the scientific community interested in the use of rats as an experimental animal model.
MATERIALS
AND METHODS
This study followed the scientific, technical, and administrative standards for health research, “Biosafety of research (Title IV, Chapter III) and biomedical research with animals (Title V)” of resolution No. 008430 of 1993, of the Colombian Ministry of Health. Laboratory procedures were governed by the biosafety standards to manage biological specimens of the Dental Research Center of Pontificia Universidad Javeriana’s Dental School from Bogotá, Colombia. This study was experimental and was approved by the Ethics and Research Committee of the Dental School and financed by the Pontificia Universidad Javeriana’s Office of the Vice-President for Research (projects 4027 and 4279).
Animals and Housing Conditions
The rats were housed under standardized conditions in ventilated cages (ONE CAGE 2000, Lab Products) under SPF conditions in the Pontificia Universidad Javeriana’s Comparative Biology-Dentistry Unit, where a Lewis colony (LEW/SsNHsd) is housed, whose founding nucleus was obtained commercially from Envigo RMS, Inc. (Indianapolis, USA). All animals were housed in groups of two, in autoclavable plastic cages (425 mm x 266 mm x 155 mm), fed ad libitum with an autoclave diet (Labdiet # 1013) and free access to purified drinking water. The light/dark cycle is 12/12, the 12 hours of light are supplied with artificial light (approximately 40 lx in the box). The temperature was 22 ± 2 °C, the relative humidity 60 ± 10 %, the environment was kept free of noise and odors. The rats were free of viruses, bacteria, and parasites listed for SPF colonies according to FELASA (43), except for Helicobacter spp.
Sixteen rats, 8 males and 8 females, 4-6 weeks of age (120-160 g of weight) were part of the study. Euthanasia was applied to the animals by means of a mixture of CO2 and air to obtain bone marrow as a source of stem cells. Cell groups of four individuals (two females and two males) were considered as a batch and the variables measured at each moment were established as a repetition until completing at least three repetitions per variable and for each batch. The characteristics of these populations were evaluated in each passage, which can provide useful information for different applications, as well as the time to obtain enough in each experiment.
Cell Culture Conditions
In all cases where the culture medium is mentioned, it refers to a-MEM (Gibcoâ 12000-014), supplemented with 2.2 g of sodium bicarbonate (Merck 106329), 10 % fetal bovine serum (Gibcoâ 16000 -044), 1 % Glutamax (Gibcoâ 35050-061), and 1 % of a commercial solution of two antibiotics (penicillin 10,000 U/ml and streptomycin 10,000 mg/ml) and an antifungal (amphotericin B 25 mg/ml) (Gibcoâ 15240-062). Serum-free medium refers to the same α-MEM culture medium, but without the supplementation with fetal calf serum. All cell centrifugation steps were performed at 300 g (2000 rpm) and 4 ºC for 5 minutes in a centrifuge (Thermo ScientificTM Heraeus Biofuge Primo R 41272826). Cell cultures were conducted in 25 cm2 culture flasks (Corning 3053) and kept at 37 ºC and 5 % CO2 in an incubator (NAPCO S400 CO2) until reaching 95 % confluence, at which time the cells were detached. The detachment of cells from the surface of the flask or trypsinization was performed with 0.5 M trypsin/EDTA (Merck 108418) for 3 minutes at 37 ºC and 5 % CO2. Cell counting was performed in a Neubauer chamber with 0.4 % Trypan Blue (Sigma T8154). Cultures were evaluated under an inverted light microscope (Leica, DMi1) and culture medium changes were performed every two days.
Obtaining Primary Cultures
A technical expert performed an aseptic dissection of the skin, muscle tissue, and periosteum of the femur and tibia bones of the hind limbs of euthanized rat, following the Ridzuan protocol (44). The next steps included the removal of bone epiphyses and medullary irrigation at one end with a culture medium-loaded syringe. The collected lavage was mechanically disintegrated with the same syringes and washed twice with culture medium. The entire cells were counted and seeded in culture flasks at a concentration of 5 x 105 cells for each flask. The primary culture (P0) was considered established when 95 % confluence was complete.
Depletion of CD45+ Populations and Sub-cultures up to P5
P0 cells were dissociated with trypsin, collected through centrifugation, counted, and incubated with 10 µl/1 x 106 cells of an antiCD45 PE (BD Pharmingen™ clone Ox-1, 554878) for 15 minutes at 4 °C to purify the CD45+ population (45). Then they were incubated for 15 minutes at 4 °C with 10 ml of a secondary antibody coupled to anti-PE magnetic beads (Miltenyi Biotech, 130048801) and this solution was passed through a magnetic separation column (Miltenyi Biotec, 120-000-472). This process allowed to retain CD45+ and collect CD45- in the eluate to corroborate the purity of the separation through flow cytometry. The CD45- population was counted and subcultured to advance towards the first passage (P1). Starting at P1, each culture that reached cell confluence was subcultured until reaching P5.
Crop Immunophenotypic Characterization
The immunophenotypic characteristics of the cultures from P0-P5 were determined through flow cytometry, using specific antibodies coupled with PE, APC and FITC fluorochromes and directed to the molecules CD45PE (554878, DB Biosciences), CD29Biot/Strep-APC (555004, DB Biosciences), CD90FITC (130-094-527, Miltenyi Biotec), CD71PE (554891, BD Biosciences and CD106PE (559229, BD Biosciences). Antibody titers in the labeled cultures were CD90: 1/100, CD29: 1/100. 1/200, CD45: 1/200, CD71: 1/100 and CD106: 1/200.
Cell Proliferation Assays
To evaluate cell proliferation, we used a colorimetric method that determines the number of viable cells in proliferation (CellTiter 96®, Promega G3580), following the manufacturer's instructions and then seeding the cells in plates (multi-wells of 96 wells—SPL Life Sciences—with a concentration of 3.2 x 103 cell/well). Evaluations took place at 12, 24, 48, 72 and 96 hours, in which the culture medium was removed from each well and a solution of the kit’s aqueous reagent in culture medium at a 1:5 ratio was added and left incubating at 37 °C for 4 hours. After that time, the optical density was read in a plate reader (Biotek ELx 800). To determine the number of cells for each absorbance value, we measured the optical density at different concentrations of adhered cells to determine a cell growth equation, through which we found the number of cells for each absorbance.
Crop Morphological Assessment
We assessed the morphological characteristics of the cultures every two days by means of an inverted microscope (Leica®, DMi1) to determine adherence to the surface of culture flasks, growth pattern, and cell confluence. After the cultures reached confluence, characterization was completed by means of Masson's Trichrome and H&E stains.
Determination of Fibroblastoid Colony-Forming Units (CFU-F)
Growth in colony-forming units for each passage measured was determined by seeding cells in 6-well multi-well plates (Corning 3506) at a concentration of 2.5 x 105 cell/well. After 21 days of culture, the cells were fixed with 10 % buffered formalin (Merck, HT50112) and stained with Gram's violet (Merck, 94448) for 30 min. The growth potential in CFU-F was verified macroscopically and the number of cells in each colony was counted microscopically. We considered CFU-F colonies with more than 30 cells. The following equation was used to calculate the efficiency in CFU-F,
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Crop Multi-Differentiation Potential
Cell cultures underwent differentiation into three lineages: osteogenic, chondrogenic, and adipogenic. Cells in each passage were seeded in triplicate in 12-well multi-well plates (Corning 3512) at a 1.5 x 105 cells/well ratio. Once they reached 60 % confluence, the culture medium was changed to induction medium. differentiation prepared by supplementing the culture medium with the reagents for induction of osteogenic (46,47), chondrogenic (25,48,49) and adipogenic (25,50) differentiation.
The osteogenic differentiation medium was prepared by supplementing the culture medium with 0.1 mM dexamethasone (Sigma-Aldrich D4902), 50 mM ascorbic acid (Sigma-Aldrich A4544) and 10 mM b glycerol phosphate (Sigma-Aldrich G9422). Cultures were maintained for 21 days with the differentiation induction medium and then washed twice with PBS (Sigma P3813), fixed with paraformaldehyde solution (Sigma Aldrich ,158127) at 4 % for 15 min, and stained with a 2 % Alizarin Red (Sigma A5533) solution and with pH 4.2 for 30 min. Finally, the cells were observed in the light microscope and the images were captured with a camera (Canon EOS REBEL T51 18-55 IS STM). To quantify osteogenic differentiation, alkaline phosphatase activity was determined using a commercial colorimetric kit (Sigma Aldrich, APO100) by following the manufacturer's instructions.
For the induction of chondrogenic differentiation, the culture medium was supplemented with 10 ng/ml human transforming growth factor β3 (TGF-β3) (Sigma-Aldrich E. coli SRP3171), 100 nM dexamethasone (Sigma-Aldrich D4902), 200 mM ascorbic acid (Sigma-Aldrich A4544), 40 mg/ml L-proline (Sigma-Aldrich 81709), 1 mM pyruvate (Sigma-Aldrich P5280), 1 mg /ml bovine serum albumin (BSA) (Sigma-Aldrich 05470), and 50 mg/ml ITS+3 Liquid Media Supplement (100x) (Sigma-Aldrich I2771). Cultures were induced with this medium for 21 days and washed twice with PBS (Sigma P3813). Then, they were fixed with a 4 % formaldehyde solution for 30 minutes, and cells were washed and visualized in a light microscope. Images were captured with a photographic camera.
Cultures with adipogenic induction were maintained for 21 days with culture medium supplemented with 1 mM dexamethasone (Sigma-Aldrich D4902), 500 mM 3-isobutyl-1-methylxanthine-IBMX (Sigma-Aldrich I5879), 5 mg/ml insulin (Sigma-Aldrich I5500), and 200 mM indomethacin (Sigma-Aldrich I7378. The dye used to identify differentiation was 0.5% Oil Red O (Sigma O1395) that was added to the cultures induced, washed, and fixed for 30 minutes. Afterwards, images were obtained with a microscope and photographic camera.
Statistical Analysis
We conducted all experiments at least three times. Descriptive data consisted of mean ± SD (p < 0.05). The Shapiro-Wilk test verified data normality, and ANOVA and Tukey’s post hoc tests determined differences between the expression of cell surface markers, cell proliferation, efficiency in the formation of colonies, and concentration of alkaline phosphatase. Student’s t test established differences in the percentage of positive cells for expression of typical stem cell markers. Data analysis was performed with SPSS software, 20.0 version 20.0 (IBM, New York, USA).
DISCUSSION
In this study, an efficient methodology for isolating, expanding, and characterizing stem cells derived from bone marrow of Lewis rats was validated, through which populations maintaining the characteristics of stemness in culture were obtained. Therefore, such cultures could be used in preclinical studies. Obtaining populations of stem cells that preserve stem immunophenotype, proliferation, and differentiation potentials during their expansion are of great interest in regenerative medicine. These characteristics allow using them in studies that seek to replicate the cell environment and its processes in vivo, in a safe and reliable way (13,51), in both cell and cell-free therapies (52).
Considering the limited regeneration potential of dental and periodontal tissues, in addition to the high incidence of pathologies that lead to their loss, translational research using stem cells and animal models seeks to find therapeutic alternatives that can be applied to humans (2) and that resolve limitations of current treatments. The characteristics displayed by bone marrow-derived stem cells, such as differentiation potential, self-renewal capacity, and immune regulation, have positioned them as a key element in gene therapy, tissue engineering, and replacement therapy applications (53). Although there are limitations in humans due to its low availability (54) and invasiveness of the process to obtaining it (55), in animal models these limitations can be reduced since the marrow tissue can be acquired from experimental colonies. Thus, the implementation of cell cultures from animal tissues becomes the basis for the establishment of reliable and predictable clinical protocols (44).
Because knowledge of the self-maintenance and differentiation mechanisms of stem cells helps to understand a variety of processes, from embryogenesis and oncogenic transformation (56), the development of methods for isolation and expansion may be the basis for dental tissue regeneration projects. They include those being conducted in our group (57). The cytokines and extracellular particles found in their conditioned media are also particularly useful for these cultures (58), which is why, in regenerative medicine, their paracrine activity has become an alternative approach for their use (59).
Bone marrow, which is especially useful because it is found in the prototype mesenchymal stem cell, has been recognized for more than three decades and has been the most studied source of stem cells (11,16,60,61). This tissue has been studied especially in mice (62) and not so much in rats (44). Rats are a model of choice in studies of physiology, behavior, and complex human diseases (63); therefore, research groups use them. In this study we obtained functional populations of stem cells derived from bone marrow, which were homogenized with the depletion of CD45+ cell subpopulations.
CD45 or leukocyte common antigen is expressed exclusively in the nucleated cells of the marrow hematopoietic fraction and is key in the activation of immune system cells (64), its expression, although at a low level, is not desirable. Therefore, we depleted our populations in the first passage, resulting in negative subcultures in the expression of this marker with an expression profile like that described in other studies (25,29,65-67). The separation method of these subpopulations did not affect the stem characteristics of the cultures since the cells were minimally manipulated. Although this depletion method had only been reported for blood cells (45,68) it shows effectiveness in the present study in the separation of subpopulations of the hematopoietic fraction that may interfere with stem cell characterization.
The immunophenotype found in the subpopulations after CD45+ depletion corresponds to that reported in other studies in which they are highly positive for CD90 (25,66), an important regulator of mesenchymal differentiation processes (69) with a proven critical role in deciding the fate of MSCs (70). It was highly positive for CD29, acting as a receptor for the union of the cell with the extracellular matrix and with other cells, whose positivity in stem cells has been related to greater cell survival when these are applied in regenerative therapies. It was also highly positive for CD146, whose expression in MSCs of multiple organs has been associated with the potential for differentiation into three lineages (71). Like in other studies, our findings show that both the cell surface protein CD71, related to intracellular iron transportation, and the cell surface protein CD106, which participates in cell immunomodulation, have extremely low positivity values (44,72).
Cellular confluence was reached approximately two weeks after starting the cultures, as has been reported in other studies (72,73). The greater the confluence, the greater the proportion of cells adhered to the surface of the culture flask with tapered and fibroblastoid morphology. This cell proliferation also showed the formation of fibroblastoid colony units, as has been reported (25,44), especially in the primary cultures. In initial subcultures, we also identified a subpopulation of polygonal cells as some have been reported somewhere else (72), which shows that these cultures correspond to heterogeneous populations that can be purified as the expansion progresses, either due to the basic characteristic of stem cells adhering to plastic or due to the selectivity of the culture medium used.
In the P2-P5 subcultures, the cell morphology was more homogeneous, most cells were elongated, fibroblastoid in appearance, aligned, and grouped. Each cell group of the latter showed a different direction, findings that are also reported in similar studies (72,73). H&E staining showed that most cells in cultures are mononucleated with large, elongated, and centrally located nuclei, as described in other studies (65,72). On the other hand, with Masson trichrome staining, type I collagen fibers were clearly visible in the extracellular spaces. Those fibers may be associated with the formation of an extracellular matrix, which has the ability of forming cell layers, which are a useful tool in tissue engineering experiments (74).
The subcultures showed that populations of this study conserved the capacity for multi-differentiation. The induced cultures experienced a change in morphology accompanied by the formation of a mineralized extracellular matrix when they were induced to the osteogenic lineage. The presence of intracytoplasmic lipid vacuoles indicated differentiation to pre-adipocytes and in the chondrogenic lineage showed the formation of a cartilage matrix. Osteogenic differentiation was also confirmed by an increase in alkaline phosphatase activity.
Studies with stem cells have shown that, despite originating from the same tissue, they have enormous variability from donor to donor (75). In this study, we used Lewis rats belonging to an inbred lineage to reduce syngeneic variability; the members of the colony are the result of mating in the first degree of consanguinity. In addition, the marrows used as stem cell source tissue were grouped into batches to include the same number of males and females in each batch, following indications of the U.S. National Institutes of Health regarding the need to include both males and females in all clinical and preclinical research (76,77). For years of research, females were not part of studies for considering that hormonal variations due to the estrous cycle could affect findings (77,78). However, studies have shown that there are no sex-related differences in neuroscience research (76) or evidence that female rats show greater inter-individual variability than males in immunological traits (77). This issue is still not completely clear so, to achieve the greatest homogeneity in the populations characterized in this study, we used tissues from both males and females.
Conducting this study allowed us to validate a methodology to culture populations of stem cells from rat bone marrow at the right densities to use in cell-based therapies or in vitro cell models, in which the collection is expected. A biological asset such as stem cells with defined characteristics that are heterogeneous, contain highly proliferative cells with the potential for differentiation into multiple lineages, and are collectable in sufficient amounts to develop tissue engineering protocols. In addition, this study provides key elements that justify studies with populations of stem cells derived from human tissues.