Isolation Of Mesenchymal Stem Cells Research Articles

Deregulation of the cyclin-dependent kinase inhibitor p27 as a putative candidate for transformation in Chlamydia trachomatis infected mesenchymal stem cells.

Several pathological conditions might cause the degradation of the cyclin-dependent kinase inhibitor (CKI) p27 and cell cycle arrest at the G1 phase, including cancers and infections. Chlamydia trachomatis (Ctr), as an obligatory intracellular pathogen, has been found to alter the fate of the cell from different aspects. In this study, we aimed to investigate the effect of Ctr infection on the expression of the important cell cycle regularity protein p27 in mesenchymal stem cells (MSCs). Isolation of MSCs from healthy human fallopian tube was confirmed by detection of the stemness markers Sox2, Nanog and Oct4 and the surface markers CD44, CD73 and CD90 by Western blotting and fluorescence-activated cell sorting analysis. The expression of p27 was downregulated at the protein level upon Ctr D infection measured by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), IF and Western blotting. Recovery of p27 in Ctr D-infected MSCs was achieved by treatment with difluoromethylornithine (DFMO). Ctr D infected MSCs were able to produce colonies in anchorage-independent soft agar assay. Ctr D infection was able to downregulate the expression of the important cell cycle regulator protein p27, which will be considered a putative candidate for transformation in Ctr D infected MSCs.

Impact of xenogenic mesenchimal stem cells secretome on a humoral component of the immune system

Objective. To determine such parameters of humoral immunity as the number of antibody-forming cells and the titer of antibodies in the blood under the action of the whole fraction of the secretome of xenogenic mesenchymal stem cells in mice with a normal immune status and mice with secondary immunodeficiency caused by the hydrocortisone acetate. Methods. The following methods were used in the study: isolation of mesenchymal stem cells from the bone marrow of cattle; culturing of isolated cells and obtaining a conditioned medium containing exometabolites with subsequent purification; modeling of secondary immunodeficiency in mice; setting up a test on mice to determine the number of antibody-forming cells in the spleen and hemagglutinin titers in blood serum; statistical data analysis. Results. A study of the impact of the secretome of xenogenic mesenchymal stem cells (MSCs) on the humoral arm of immunity in mice with a normal immune status showed a significant increase in the antibody-forming cells count compared with the control by 3.4-8.8 times when administered intramuscularly and by 4.2-5.4 times when administered subcutaneously (at the same time, in the group of the reference drug, the increase was four times), as well as an increase in the titer of hemagglutinins concerning the control by 25-95% when administered intramuscularly and 32-52% when administered subcutaneously (the reference drug Thymalin was at the control level). With intramuscular administration, a clear relationship was observed between the concentration of MSC secretome and the biological effect (high significant positive correlation: ρ = 0.99, p ≤ 0.05 for calculating AFC; ρ = 0.97, p ≤ 0.05 for HA titer). Lethality in the group of animals what administered only hydrocortisone acetate (HCA, positive control) was 100%. The use of the mesenchymal stem cells secretome increased the survival of animals by 50% by stimulating the formation of the required number of antibody-forming cells and antibody titer, except for the subcutaneous route of administration (at the level of immunized control, animals with a normal immune status). The reference drug showed a result at a level significantly lower than the immunized control. The antibody titer with the subcutaneous route of administration of secretion of MSCs was significantly lower than the immunized control but significantly higher concerning the reference drug. Conclusion. The administration of the secretome of xenogenic mesenchymal stem cells stimulated the humoral arm of immunity as same in mice with normal immune status as in mice with secondary immunodeficiency. The data obtained supplement the information on the introduction of live mesenchymal stem cells. Live allogeneic MSCs have a suppressive effect on B cells, while xenogenic MSCs cause a response on themselves. The secretome of xenogenic MSCs does not contain surface immunogenic molecules that are carried by living cells, but nevertheless increases the activity of the humoral component of immunity. The mechanisms of this effect require further study.

Scalable mesenchymal stem cell enrichment from bone marrow aspirate using deterministic lateral displacement (DLD) microfluidic sorting.

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Isolation and Cultivation of Adipose-Derived Mesenchymal Stem Cells Originating from the Infrapatellar Fat Pad Differentiated with Blood Products: Method and Protocol

Adipose-derived mesenchymal stem cells (ASCs) are a promising source for clinical application in regenerative orthopedics. ASCs derived from the infra-patellar fat pad (IFP)-a distinct adipose structure in the knee-show superior regenerative potential compared to subcutaneous-fat-derived cells. Furthermore, it has been shown that blood products enhance ASCs' viability. A major challenge for clinical translation of both ASCs and blood products is the low comparability of obtained data due to non-standardized harvesting, isolation and preparation methods. The aim of this method-paper is to provide reproducible protocols to help standardize basic research in the field to build a sound basis for clinical translation with an emphasize on practicability. The presented protocols include (i) ASC isolation from the IFP, (ii) blood product preparation and (iii) ASC incubation with blood products.

Isolation and Identification of Bone Marrow Mesenchymal Stem Cells from Forest Musk Deer

The forest musk deer (Moschus berezovskii) is an endangered animal that produces musk that is utilized for medical applications worldwide, and this species primarily lives in China. Animal-derived musk can be employed as an important ingredient in Chinese medicine. To investigate the properties of bone marrow mesenchymal stem cells (MSCs) obtained from the bone marrow of forest deer for future application, MSCs were isolated and cultivated in vitro. The properties and differentiation of these cells were assessed at the cellular and gene levels. The results show that 81,533 expressed genes were detected by RNA sequencing, and marker genes of MSCs were expressed in the cells. Karyotype analysis of the cells determined the karyotype to be normal, and marker proteins of MSCs were observed to be expressed in the cell membranes. Cells were differentiated into osteoblasts, adipocytes, and chondroblasts. The expression of genes related to osteoblasts, adipocytes, and chondroblasts was observed to be increased. The results of this study demonstrate that the properties of the cells isolated from bone marrow were in keeping with the characteristics of MSCs, providing a possible basis for future research.

Current advances in stem cell therapy in the treatment of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease related to the central nervous system (CNS) with a significant global burden. In this illness, the immune system plays an essential role in its pathophysiology and progression. The currently available treatments are not recognized as curable options and, at best, might slow the progression of MS injuries to the CNS. However, stem celltreatment has provided a new avenue for treating MS.Stem cells may enhance CNS healing and regulate immunological responses. Likewise, stem cells can come from various sources, including adipose, neuronal, bone marrow, and embryonic tissues. Choosing the optimal cell source for stem cell therapy is still a difficult verdict. A type of stem cell known as mesenchymal stem cells (MSCs) is obtainable from different sources and has a strong immunomodulatory impact on the immune system. According to mounting data, the umbilical cord and adipose tissue may serve as appropriate sources for the isolation of MSCs. Human amniotic epithelial cells (hAECs), as novel stem cellsources with immune-regulatory effects, regenerative properties, and decreased antigenicity, can also be thought of as a new upcoming contender for MS treatment. Overall, the administration of stem cells in different sets of animal and clinical trials has shown immunomodulatory and neuroprotective results. Therefore, this review aims to discuss the different types of stem cells by focusing on MSCs and their mechanisms, which can be used to treat and improve the outcomes of MS disease.

Simple Procedure for the Isolation of Mesenchymal Stem Cells from Different Parts of the Human Umbilical Cord

The umbilical cord and placenta are both sources of mesenchymal stem cells (MSCs) that are promising for cell-based therapy. Furthermore, compared to other MSCs sources, they are easy to obtain with no invasive procedures. This study presents an adapted method for stem cell isolation from three different parts of the human umbilical cord, including Wharton’s jelly (WJ), cord lining (CL), and cord-placenta junction (CPJ). The isolation consists of sample preparation, tissue dissection into distinct anatomical regions, mincing and enzyme digestion, and explant culturing. In addition, we monitored when the cells migrated from the explant to the bottom of the cell culture dish and passed the cells after they became confluent. This study found that WJ cells were the first to reach confluence at Passage 0 (P0). In contrast, CL cells needed the longest time to get confluence at P0 but displayed faster cell growth after subsequent passages (P1-P2). In addition, CPJ cells showed growth retardation after P1 and P2. Altogether, we could extract the MSCs from umbilical cord tissue explants by using DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin as general cell culture medium and omitting the use of gentamicin. However, the MSCs may need a more complex specified medium for optimum cell regeneration for further cell expansion.Keywords: mesenchymal stem cells, umbilical cord, Wharton’s jelly, cord lining, cord-placenta junction.

Low Oxygen Tension Inhibits Senescence in Bone Marrow Mesenchymal Stem Cells (BMSCs)

Background: Stem cells have been used in regenerative medicine but are so few in the body that they require cell culture. Stem cell culture was performed under normal oxygen tension and passage was carried out until the number of cells was sufficient for therapy. Stem cell cultures under normal oxygen tension do not match the stem cell microenvironment, which can lead to premature senescence. This study aims to determine the association of low oxygen tension with premature senescence of mesenchymal stem cells (MSCs) through inhibition of p21 expression by HIF-1a. Methode: The research method used rabbit bone marrow in New Zealand as a source of MSCs. The results of isolation of MSCs were divided into two groups for cultured on normal and low oxygen tension until 10 passages. Cells were identified using flowcytometry for cd105 and cd34. At early and late passage, the expression of p21 and HIF-1a was examined using immunofluorescence while senescence was examined using β-galactosidase assay. Results: The results showed that in low oxygen cultures HIF-1a expression increased significantly (p <0.05) while p21 expression decreased significantly (p <0.05) as did the β-galactosidase assay. Conclusions: The conclusion of this research is low oxygen tension culture able to decrease premature senescence culture of invitro stem cells mesenchymal through obstacles p21 by HIF-1a.
 Keywords: p21 expression, HIF-1a expression, late passage, premature senescence.

Optimizing aseptic and serum milieu for the isolation of human whole umbilical cord tissue-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have become an attractive tool for tissue engineering and targets in clinical transplantation due to their regeneration potential and immune-suppressive capacity. The human umbilical cord, which is discarded at birth, can provide an inexhaustible source of stem cells for therapy. They are reported to contain immune privilege cells which may be suitable for allogenic-based therapies. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for the generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs.ResultsMSCs were isolated from the umbilical cord by explants and enzymatic digestion and cultured in the appropriate growth medium resulted in the propagation of more than 1X 108 cells within 15 days from the single umbilical cord.ConclusionThe isolation efficiency, cell yield, colony-forming unit fibroblast (CFU-F), growth kinetics, phenotypic characteristics of UCMSCs were determined.

Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs

BackgroundUtilization of autologous stem cells has been proposed for the treatment of anal incontinence despite a lack of understanding of their mechanism of action and of the physiological healing process of anal sphincters after injury.AimsWe aim to develop a technique allowing isolation and further study of local mesenchymal stem cells, directly from anal canal transition zone in pig.MethodsAnal canal was resected “en bloc” from two young pigs and further microdissected. The anal canal transition zone was washed and digested with 0.1% type I collagenase for 45 min at 37 °C. The isolated cells were plated on dishes in mesenchymal stem cell medium and trypsinized when confluent. Cells were further used for flow cytometry analysis and differentiation assays.ResultsThe anal canal transition zone localization was confirmed with H&E staining. Following culture, cells exhibited a typical “fibroblast-like” morphology typical of stem cells. Isolated cells were positive for CD90 and CD44 but negative for CD14, CD34, CD45, CD105, CD106, and SLA-DR. Following incubation with specific differentiation medium, isolated cells differentiated into adipocytes, osteoblasts, and chondrocytes, confirming in vitro multipotency.ConclusionsHerein, we report for the first time the presence of mesenchymal stem cells in the anal canal transition zone in pigs and the feasibility of their isolation. This preliminary study opens the path to the isolation of human anal canal transition zone mesenchymal stem cells that might be used to study sphincters healing and to treat anal incontinence.

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45-CD34-, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

Umbilical cord blood: an undervalued and underutilized resource in allogeneic hematopoietic stem cell transplant and novel cell therapy applications.

The purpose of this review is to primarily discuss the unwarranted decline in the use of umbilical cord blood (UCB) as a source of donor hematopoietic stem cells (HSC) for hematopoietic cell transplantation (HCT) and the resulting important implications in addressing healthcare inequities, and secondly to highlight the incredible potential of UCB and related birthing tissues for the development of a broad range of therapies to treat human disease including but not limited to oncology, neurologic, cardiac, orthopedic and immunologic conditions. When current best practices are followed, unrelated donor umbilical cord blood transplant (CBT) can provide superior quality of life-related survival compared to other allogeneic HSC donor sources (sibling, matched or mismatched unrelated, and haploidentical) through decreased risks of relapse and chronic graft vs. host disease. Current best practices include improved UCB donor selection criteria with consideration of higher resolution human leukocyte antigen (HLA) typing and CD34+ cell dose, availability of newer myeloablative but reduced toxicity conditioning regimens, and rigorous supportive care in the early posttransplant period with monitoring for known complications, especially related to viral and other infections that may require intervention. Emerging best practice may include the use of ex vivo expanded single-unit CBT rather than double-unit CBT (dCBT) or 'haplo-cord' transplant, and the incorporation of posttransplant cyclophosphamide as with haploidentical transplant and/or incorporation of novel posttransplant therapies to reduce the risk of relapse, such as NK cell adoptive transfer. Novel, non-HCT uses of UCB and birthing tissue include the production of UCB-derived immune effector cell therapies such as unmodified NK cells, chimeric antigen receptor-natural killer cells and immune T-cell populations, the isolation of mesenchymal stem cells for immune modulatory treatments and derivation of induced pluripotent stem cells haplobanks for regenerative medicine development and population studies to facilitate exploration of drug development through functional genomics. The potential of allogeneic UCB for HCT and novel cell-based therapies is undervalued and underutilized. The inventory of high-quality UCB units available from public cord blood banks (CBB) should be expanding rather than contracting in order to address ongoing healthcare inequities and to maintain a valuable source of cellular starting material for cell and gene therapies and regenerative medicine approaches. The expertise in Good Manufacturing Practice-grade manufacturing provided by CBB should be supported to effectively partner with groups developing UCB for novel cell-based therapies.

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco's modified Eagle medium (HG-DMEM), β-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific β-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

Ficoll density gradient sedimentation isolation of pelage hair follicle mesenchymal stem cells from adult mouse back skin: a novel method for hair follicle mesenchymal stem cells isolation

BackgroundHair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring.MethodsTwo-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining.ResultsIsolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes.ConclusionA large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle.

Therapeutic and clinical propositions of Mesenchymal Stem Cells(MSCs) focusing on recent trial updates

The mesenchymal stroma harbours considerable population of stem cell-like cells with differentiation and self-renewal abilities that originate from several sources like amniotic fluid, Wharton’s jelly, umbilical cord etc. Mesenchymal Stem Cells (MSCs) are most commonly found in the perivascular niche. This multipotent progenitor cells have the ability to differentiate into mesodermal cell types such as adipocytes, chondrocytes and osteocytes. MSCs can also exert significant immunosuppressive and anti-inflammatory effects by interacting with lymphocytes from both innate and adaptive immune system. MSCs of fetal origins can go through same processes as MSCs derived from elsewhere. Before senescence, they make more cell divisions than adult MSCs of bone marrow or adipose tissue. The propitious properties of mesenchymal stem cells (MSCs), such as their ability to differentiate into diverse cell lineages and their regenerative properties, have intrigued researchers, whose work has provided fascinating perspectives on cell-based therapies for wide range of diseases. MSCs have a high potential for replication in vitro. International Society for Cellular Therapy (ISCT)-based MSC isolation has resulted in heterogeneous, nonclonal stromal cell cultures containing stem cells with various multipotent characteristics, committed progenitors, and differentiated cells. Nonclonal stromal cultures which are derived from bone marrow and other tissues are presently used as sources of putative MSCs for therapeutic aims. Also, MSCs have potential to endogenously repair as well as reduce the inflammatory responses that may lead to decreased morbidity and mortality rate of COVID-19. Herein, the MSC-based clinical prospects, clarifies, the recent clinical findings, therapeutic effects of MSC and clinical trials.

Isolation, culture and identification of SD rat bone marrow mesenchymal stem cells in tissue engineering

Seed cells are an important part of bone tissue engineering. In this study, we investigated methods for the isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs from SD rats were isolated and cultured using the whole bone marrow adherent method. Morphological observation and flow cytometry were used for the detection of cell surface markers. The osteogenic and adipogenic differentiation of cultured cells was induced and evaluated. BMSCs could be effectively isolated and purified using the whole bone marrow adherent method. The primary BMSCs cultured in vitro formed spindle-shaped, spiral and vigorous cell colonies. The flow cytometry results showed that CD90 was highly expressed on the cell surface, but CD34 and CD45 were not expressed. After culture in osteogenic and adipogenic induction medium, positive Alizarin Red and Oil Red O staining was observed, indicating the potential for multidirectional differentiation. The whole bone marrow adherent method is simple and practical, which makes the isolation, culture and amplification of BMSCs in vitro easy, and the method has little effect on cell activity and is an effective method for obtaining high-purity BMSCs.

Isolation and identification of Murine Mesenchymal Stem Cells from Bone Marrow.

Background: Mesenchymal stem cells (MSCs) are beneficial for cell and gene therapy approaches. These cells are highly potent in producing various cells, including adipocytes, osteoblasts, hematopoietic cells, etc., particularly bone marrow-derived. High proliferation and regenerative capacity are important phenotypes of MSCs which contribute to the recovery process at the damaged sites. Various techniques have been used, which have inconvenient effects on stromal cell properties. The present study evaluates the stem cell markers in the isolated cells from the Murine mouse bone marrow. Methodology: The murine mesenchymal stem cells (mMSCs) were isolated from the bone marrow of murine Balb/c mice using a simple in vitro culture protocol. Trypan blue exclusion assay was performed for viable cell count, and cellular morphology was checked using an inverted microscope. The immunofluorescence staining was done to analyze the immunophenotypic features of mMSCs by treating cells with CD44, CD90, and CD45 fluorescent antibodies. Results: mMSCs with spindle-like cell appearance were attained within three weeks. Immunofluorescence staining of isolated mMSCs displayed a statistically significant (p<0.001) number of CD44 and CD90 positive cells. Alternatively, an insignificant count of CD45 labeled cells was found. Conclusion: A uniform mMSC population was achieved with high propagation capability in lower passages through regular replacement of medium and reduced trypsinization time. In addition, mMSCs were strongly expressed MSCs positive markers, including CD44 and CD90.