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Research Article | DOI: https://doi.org/10.31579/IJBR-2021/024
*Corresponding Author: Azab Elsayed Azab, Department of Physiology, Faculty of Medicine, Sabratha University, Libya
Citation: Marwan T. M. Abofila1&, Azab Elsayed Azab, Imam U. M, M. H. Ng, Ramasamy R, Chen H. C, Ganabadi S. (2021) Heart Failure: Symptoms, Diagnosis, Prevention and Treatment with Special Reference to African-Americans, International J. of Biomed Research 1(4); DOI: 10.31579/IJBR-2021/024
Copyright: © 2021 Prabir Mandal, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 02 June 2021 | Accepted: 05 June 2021 | Published: 16 June 2021
Keywords: mesenchymal stem cells, bone marrow, new zealand white rabbit, cell morphology, multipotency, immunophenotyping
Background: Stem cells are defined as cells that can self-renew indefinitely and able to differentiate into various mature cells when induced appropriately. It have many other properties bring attention to use in regenerative medicine. Stem cell therapy has attracted much interest in this 21st century, not only because of the controversy surrounding the ethics involving pluripotent stem cells, but their potential for clinical use.
Objectives: The aim of this study was to isolate and characterize mesenchymal stem cells from bone marrow of New Zealand white rabbits by its morphological, multipotential differentiation and immunophenotypical analysis.
Materials and Methods: Three rabbits were euthanized with pentobarbital, an incision was made through the skin at thigh region and all muscles related to femoral bone were removed. The two epiphysis ends were cut ant the bone marrow was flushed to be cultured for series of passages.
Results: The bone marrow cells were shown to adhere to the plastic surface and started to form fibroblastic-like colonies after 3 to 7 days of initial seeding. Further characterization was conducted using cells from passage two onward by analyzing their surface protein expression and ability to differentiate into mesodermal lineages under a relevant inductive condition. Flow cytometer analysis showed that the adherent bone marrow cells were expressing CD44 and CD90 but not CD34 which is a standard profile of mesenchymal stem cells. Besides, the bone marrow cells which were subjected to adipogenic and osteogenic differentiation exhibited differentiation into adipocytes and osteoblasts when cultured in appropriate inductive differentiation media.
Conclusion: Based on our observation, the bone marrow adherent cells from New Zealand white rabbits had reflected common mesenchymal stem cells characteristics which have been confirmed via morphological, multipotential ability and immunophenotyping analysis.
Stem cells are defined as cells that can self-renew indefinitely and able to differentiate into various mature cells when induced appropriately [1]. It have many other properties bring attention to use in regenerative medicine. Stem cell therapy has attracted much interest in this 21st century, not only because of the controversy surrounding the ethics involving pluripotent stem cells, but their potential for clinical use [2].
Stem cells are classified according to their sources into two main types, the embryonic and non-embryonic. Embryonic stem cells (ESC) are pluripotent and can differentiate into all germ layers [3]. Non-embryonic stem cells (Non-ESC) can be sub-classified into fetal stem cells (FSCs) and adult stem cells (ASCs) [2]. Both of them are multipotential. Their potential to differentiate into different cell types appears to be more limited [3]. The capacity of these cells for potency (power) and relative ease to isolate and expand are valuable properties for regenerative medicine [4].
Mesenchymal stem cells have been shown to adhere to cell culture flask and exhibit fibroblastic-like shape. Many studies have been demonstrated the effects of different culture protocols on the cell phenotype. The reports show little and no significant differences among the cells isolated by any protocol [5-7].
Differentiation is the process by which matured cells change to a specialized type. During differentiation, certain genes are turned on and become activated while others are switched off and become inactivated; a complex process tightly regulated, resulting in cell development of specific structures, which perform certain functions. Cultured cells can be made to differentiate into exclusive lineages by providing Selective media components that can be identified by histochemical staining and quantified by quantitative Real-time polymerase chain reaction (RT-PCR) [8]. The standard test to confirm the mesenchymal stem cells is differentiation of the cells into other specific cells such as osteoblasts, adipocytes, chondrocytes, myocytes and neurons. MSCS have been seen to even differentiate into neuron-like cells. The process of differentiation normally will occur with the aid of influencing factors and it is considered the test for multipotency of MSCs [9].
Immunophenotyping is using a flow cytometry technique to enable identification of specific cell types from complex biological samples according to the cell surface antigen expression. Mesenchymal stem cells can be identified based on the expression of specific proteins called surface antigen phenotype of mesenchymal stem cell markers. Some of these markers are present on undifferentiated MSCs and disappear during differentiation [10].
The aim of this study was to isolate and characterize mesenchymal stem cells from bone marrow of New Zealand white rabbits by its morphological, multipotential differentiation and immunophenotypical analysis.
3.1 Experimental animals
The use of animals were approved and in accordance to the guide by the Animal Care and Use Committee (ACUC), Faculty of Veterinary Medicine, University Putra Malaysia (Ref.UPM/FRV/PS/3.2.1.551/ AUP-R94). Three New Zealand White rabbits, 3 to 4 months old, weighing 1.3 ± 0.3 kg were used in this experiment to take stem cells derived bone marrow. All rabbits were healthy based on physical and blood profile examinations. , on 9th April 2010
3.2 Isolation of mesenchymal stem cells from rabbit bone marrow (BM-MSCs)
The isolation of MSCs was performed on euthanized rabbits as illustrated by Braga-Silva et al. [11]. This included anesthetizing the rabbits with ketamine-xylazine and subsequently euthanizing them with sodium pentobarbital (Dolethal). An incision was then made through the skin on the cranial thigh region and all muscles attached to the femur were removed to allow for a brief immersion of the femoral bone in 70% alcohol. The femoral bone was later placed in a 50 ml falcon tube containing media and both ends of the epiphysis cut using a bone cutter. Finally, bone marrow was flushed out into a 15ml falcon tube with 5ml media.
The collected bone marrow was immediately mixed with 5 ml of 83% Dulbecco’s Modified Eagle’s Medium Ham’s F12 (DMEM F12) that contained high glucose supplemented with 15
4.1 Characterization of Rabbit BM-MSCs
The isolated cells were pre-characterized by their morphology, differentiation capacity and expressed CD markers to ensure that the isolated cells were MSCs in nature.
4.1.1 Morphology
In this study, the isolated cells suspended in media were regularly rounded in shape, evenly distributing, albeit occasional cells clumping (Figure .1). Cells adhered to tissue culture flasks after 3 days -7 days of seeding, and it exhibited a fibroblast-like shape. The population became more homogeneous after subsequent passages (Figures .2-4).
4.2 Differentiation Potential
Pellet culture of rabbit BM-MSCs in adipogenesis media after 21 days showed positive deposition of fat droplets by using Oil Red Stain and the cells’ nuclei were stained purple with hematoxylin stain. This indicated the differentiation of MSCs to adipocytes containing the intra-cytoplasmic lipid vacuoles (fat droplet). Meanwhile the cultured cells of rabbit BM-MSCs in control media did not stain positive for Oil Red Stain (Figure .5).
The rabbit BMSCs cultured in osteogenesis media for 21 days showed positive deposition of calcium as detected by Alizarin Red S stain. This indicated the differentiation of MSCs to osteocytes. While the rabbit BM-MSCs cultured in control media did not stain positively for Alizarin Red S Stain (Figure 6). Overall, the rabbit BM-MSCs showed multi-potential ability.
4.1.3 Immunophenotyping of Stem Cells
The second passage of rabbit BM-MSCs was utilized for detection of the cell surface epitopes. Those cells were positive in the expression of MSCs marker related to cell adhesion CD44 (Pgp-1, HCAM) (Figure 3.7) and
multipotency CD90 (Thy-1) (Figure 8). While the same cells showed negative in the expression of hematopoietic stem cells marker CD34 (Figure 3.9), they also showed negative even in the presence of second (2ed) antibody only (negative control), (FITC-conjugated anti-mouse IgG1 antibody) (Figure .10).
The Morphological Characteristic: the present study demonstrated that BM-MSCs adhered to tissue culture flasks after 3 to 7 days of seeding and exhibited fibroblast-like shapes. BM-MSCs were isolated based on their adherence to tissue culture flasks and survival through the cultivation period. These may be possible because they maintained their proliferative capacity, as reported previously [18, 19]. Two populations of cells in primary passage (P0) were identified including small round cell (non-adherent cells) and spindle-shaped stromal stem cells (adherent cells). The small rounded cells decreased in size rapidly and disappeared with repeated passages in a culture of regular media, while the fibroblastic cells maintained their proliferative characteristics similar to those in the primary passage (P0). The cells required up to 14 days to reach 90% confluence, at the first passage (P1), and showed complete confluence at day 5 of subculture. In the second passage (P2), cells achieved confluence on day 2, and by the third and fourth passages (P3 and P4), cells became morphologically homogeneous with more than 95% showing fibroblastic shapes. All these heterogeneous and homogeneous changes in morphology observed were in agreement with previous reports (12, 18-20].
The Immunophenotyping Characteristic: the present result demonstrated that BM-MSCs derived from rabbits exhibited a similar expression profile as MSC surface markers (cell adhesion marker CD44 and multipotency marker CD90) and lacked hematopoietic markers (CD34). However, previous studies investigating MSCs isolated from various tissues have indicated that cell surface phenotype can vary from that of BM-MSCs [21, 22], most notably using a panel of antibodies used to define MSCs [23]. In BM-MSCs, the surface markers have clearly been defined, the success of which has depended largely on their ability to adhere to tissue culture flasks that separate them from cells of hematopoietic stem cells [24].
The Multipotential Character: the differentiation potential of BM-MSCs derived using two different techniques (by way of anesthesia or euthanasia) demonstrated similar differentiation capacities in comparison with most previous studies [20, 25]. The isolated cells incubated in adipogenic induction medium were able to differentiate into adipocytes with accumulation of intracellular lipid droplets detected by staining with Oil Red O. This result is consistent with previous reports [20, 25, and 26]. Also, BM-MSCs incubated in an osteogenic induction medium were able to differentiate into osteocytes with intracellular calcium deposit as demonstrated by red coloration of Alizarin S, in agreement with what has been reported previously [20, 27, 28]. Other studies have reported that BM-MSCs are multipotent cells that can differentiate into different cell types, including adipocytes and chondrocytes [29]. Besides that, these cells have the ability to Trans-differentiate into cells of endoderm and ectoderm lineages such as hepatocytes and neurons [30].
Overall, The confirmation and identification of MSCs through morphology, multipotency and immunophenotyping characteristics have proven that the isolated cells from rabbit bone marrow were MSCs in nature, which is consistent with reported findings [12, 18-20, 23-26].
In general, the morphological, multipotency and immunophenotypical characteristics for the isolated MSCs from euthanized rabbit bone marrow have proven that those cells were stem cell in nature. The isolated MSCs from euthanized rabbit bone marrow illustrated similarities in morphological characteristics and multipotential ability compared with other research work which were isolated MSCs from anesthetized rabbit, even though variations existed in the sample harvesting, materials and required skills in the two methods. Euthanized rabbits were subsequently used as the method of choice for harvesting the bone marrow cells. Further comparative study should be made to study cell viability and proliferation capacity between bone marrow derived stem cells from anaesthetized and euthanized animals, moreover study the advantage and disadvantage for each method.
Researchers appreciate the effort and the assistance that was offered by the University Putra Malaysia and University Kebangsaan Malaysia as well as Zawia University and Sabratha University - Libya. In addition, the researchers are grateful to Dr. Rajash Ramasamy and Dr. Angel Ng for their help, guidance, and inspiration in carrying out this work". Instead of the way it was written in this article.
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