• bone marrow
• cell proliferation
• critical quality attributes
• immune suppression
• mesenchymal stem cells
• stromal cells

• Animals
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/metabolism
• Mice
• Cell Differentiation
• Translational Research, Biomedical
• Humans
• Pelvis
• Bone and Bones/cytology
• Bone Marrow Cells/cytology
• Cell Proliferation
• Mice, Inbred C57BL
• Nitric Oxide Synthase Type II/metabolism

• R01 HL144089 NHLBI NIH HHS
• R24 OD018254 NIH HHS
• OD018254 and HL144089 NIH HHS

• aortic aneurysm
• extracellular vesicles
• mesenchymal stem cells

• Humans
• Extracellular Vesicles
• Aortic Aneurysm, Thoracic/diagnostic imaging
• Aortic Aneurysm, Thoracic/surgery
• Mesenchymal Stem Cells

• R01 HL169390 NHLBI NIH HHS
• R56 HL161454 NHLBI NIH HHS

• Allograft
• Bilayer bioscaffold
• Decellularized whole osteochondral
• Mesenchymal stem cells
• Subcutaneous implantation

• Animals
• Rabbits
• Tissue Engineering/methods
• Cartilage, Articular
• Chondrocytes
• Mesenchymal Stem Cells
• Cell Differentiation
• Tissue Scaffolds

• M2 macrophages
• amniotic membrane
• biomaterials
• monocyte
• skin regeneration
• tissue engineering
• wound healing

• Amnion
• Animals
• Humans
• Macrophages
• Mesenchymal Stem Cell Transplantation
• Mesenchymal Stem Cells/metabolism
• Models, Animal
• Skin
• Wound Healing

• 97-02-87-33265 Iran University of Medical Sciences

• Animals
• DNA Damage
• Metal Nanoparticles/toxicity
• NF-kappa B/metabolism
• Nanoparticles/toxicity
• Rats
• Titanium/toxicity

• 95/2-3/6 Tabriz University of Medical Sciences

• Characterization
• mesenchymal stem cells
• mice
• shoulder
• subacromial bursal tissue

The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion.

In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes).

Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1⁺CD44⁺F4/80⁻ mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays.

Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.

The online version contains supplementary material available at 10.1007/s13770-021-00412-6.

• Bone marrow
• Isolation
• Liposomal clodronate
• Macrophages
• Mesenchymal stem cells
• Mouse

• Differentiation
• Hepatic stem/progenitors
• Liver fibrosis
• Mesenchymal stem cells
• Regeneration

Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro.

All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion.

The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 10⁷cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12µM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology.

Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

• Bone marrow
• Cell culture techniques
• In vitro techniques
• Mesenchymal stem cells
• Mice

• cytokine/chemokine secretion
• human cells
• immunophenotype
• in vitro modeling
• mice
• microarc calcium phosphate coating
• motility
• osteogenic differentiation
• subcutaneous behavior of implants
• surface morphology

Type 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy.

hTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.

Immunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.

At 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.

• Cellular biology
• Glucose uptake
• Hyperglycemia
• Insulin resistance
• Obese
• Orthopaedics
• Tendon
• Tenocyte
• Tumor necrosis factor-alpha (TNF-α)
• Type II diabetes

• exosomes
• mesenchymal stem cell (MSC)
• regenerative medicine
• traumatic brain injury (TBI)

Spleen stromal lines which support in vitro hematopoiesis are investigated for their lineage origin and hematopoietic support function in vivo. Marker expression and gene profiling identify a lineage relationship with mesenchymal stem cells and perivascular reticular cells described recently in bone marrow. Stromal lines commonly express Cxcl12, Pdgfra and Pdgfr typical of bone marrow derived perivascular reticular cells but reflect a unique cell type in terms of other gene and marker expression. Their classification as osteoprogenitors is confirmed through ability to undergo osteogenic, but not adipogenic or chondrogenic differentiation. Some stromal lines were shown to form ectopic niches for HSCs following engraftment under the kidney capsule of NOD/SCID mice. The presence of myeloid cells and a higher representation of a specific dendritic-like cell type over other myeloid cells within grafts was consistent with previous in vitro evidence of hematopoietic support capacity. These studies reinforce the role of perivascular/perisinusoidal reticular cells in hematopoiesis and implicate such cells as niches for hematopoiesis in spleen.

• Cartilage
• Comparison of wavelengths
• Low level light therapy
• Mesenchymal stem cells, bone
• Photobiomodulation

• Alkaline Phosphatase/genetics
• Alkaline Phosphatase/metabolism
• Animals
• Bone and Bones/cytology
• Cartilage/cytology
• Cell Differentiation/radiation effects
• Cell Lineage/radiation effects
• Cell Proliferation/radiation effects
• Cell Shape/radiation effects
• Cells, Cultured
• Chondrogenesis/genetics
• Chondrogenesis/radiation effects
• Collagen Type I/genetics
• Collagen Type I/metabolism
• Collagen Type II/genetics
• Collagen Type II/metabolism
• Gene Expression Regulation/radiation effects
• Lasers
• Low-Level Light Therapy
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/radiation effects
• Osteogenesis/genetics
• Osteogenesis/radiation effects
• Rabbits

• R01 AI050875 NIAID NIH HHS
• R21 AI121700 NIAID NIH HHS
• R01AI050875 National Institute of Allergy and Infectious Diseases
• R21AI121700 National Institute of Allergy and Infectious Diseases

• BMSC
• Ectopic bone
• Osteoblast
• Stem cells
• bFGF

• Deanship of Scientific Research, King Faisal University
• Novo Nordisk Foundation Center for Basic Metabolic Research
• Lundbeckfonden

Mesenchymal stem or stromal cells (MSCs) are identified as sources of pluripotent stem cells with varying degrees of plasticity. Endothelial progenitor cells (EPCs) originate from either bone marrow (BM) or peripheral blood and can mature into cells that line the lumen of blood vessels. MSC and EPC therapies exhibit promising results in a variety of diseases. The current study described the simultaneous isolation of EPCs and MSCs from murine BM using a straightforward approach. The method is based on differences in attachment time and trypsin sensitivity of MSCs and EPCs. The proposed method revealed characteristics of isolated cells. Isolated MSCs were positive for cell surface markers, cluster of differentiation (CD)29, CD44 and stem cell antigen-1 (Sca-1), and negative for hematopoietic surface markers, CD45 and CD11b. Isolated EPCs were positive for Sca-1 and vascular endothelial growth factor receptor 2 and CD133. The results indicate that the proposed method ensured simultaneous isolation of homogenous populations of MSCs and EPCs from murine BM.

• endothelial progenitor cells
• isolation
• mesenchymal stem or stromal cells
• murine bone marrow
• plastic adherence

• amputation
• clinical applications
• gene therapy
• limb regeneration
• stem cell therapy
• tissue engineering

• Human bone marrow derived mesenchymal stem cells
• Lipopolysaccharide
• Shigella
• Toll-like receptor 4

• Bioelectromagnetism
• DNA dielectic moment
• DNA electromagnetic properties

In vitro culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) has been hampered because of the low yield of MSCs during isolation and the contamination of hematopoietic cells during expansion. The lack of specific mouse BM-MSC markers increases the difficulty. Several techniques have been reported to improve the purity and in vitro growth of mouse BM-MSCs. However, systematic report on comparison of characteristics in primary BM-MSCs between different culture conditions is rare. Here, we studied the effects of oxygen concentrations and initial medium replacement intervals, along with cell passages, on mouse BM-MSCs isolated with differential adhesion method. BM-MSCs exhibited elevated proliferative and clonogenic abilities in 5% oxygen compared with 10% and 21% oxygen, as well as a better expression of the MSC marker Sca-1. Adipogenic and osteogenetic differentiation of BM-MSCs can be observed in both 21% and 5% oxygen. Adipogenic differentiation appeared stronger under normoxia conditions. BM-MSCs showed increased proliferative capacity and adipogenic/osteogenetic differentiation potential when initial medium replacement interval was 4 days compared with 1 day. As passage number increased, cells were more MSC-like in morphology and in expression of surface markers (positive for CD29, CD44, and Sca-1 and negative for CD11b, CD19, and CD45). These data provide new insight into optimizing the culture method and understanding the biological characteristics of mouse BM-MSCs during in vitro expansion.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

• Dermal fibroblasts
• Foxn1
• Skin
• TGFβ
• Wound healing

• Animals
• Culture Media, Conditioned
• Disease Models, Animal
• Fibroblasts/cytology
• Fibroblasts/metabolism
• Forkhead Transcription Factors/deficiency
• Keratinocytes/cytology
• Keratinocytes/metabolism
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Skin/cytology
• Skin/metabolism
• Transforming Growth Factor beta1/biosynthesis
• Transforming Growth Factor beta1/genetics
• Transforming Growth Factor beta1/pharmacology
• Transforming Growth Factor beta3/biosynthesis
• Transforming Growth Factor beta3/genetics
• Transforming Growth Factor beta3/pharmacology

• KNOW (Leading National Research Centre) Scientific Consortium ’’Healthy Animal – Safe Food”, decision of Ministry of Science and Higher Education
• Institutional Grant of Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn

Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages.

In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BM- MSCs and BlCs cultures at days 7, 14, and 21.

qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points.

Characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.

• Blastema Cells
• Mesenchymal Stem Cells
• Osteogenesis
• Regeneration

• Msx gene transduction
• animal model
• blastema cells
• bone
• bone differentiation
• bone marrow-derived mesenchymal stem cells
• differentiation
• limb regeneration
• mesenchymal stem cells (MSCs)
• regeneration

• Bone-marrow-derived mesenchymal stem cells
• Hematopoietic cell E-/L-selectin ligand
• Ischemic kidney injury
• Macrophage polarization

• Animals
• Cell Movement/physiology
• Cell Polarity
• Cell Transdifferentiation/physiology
• Endothelial Cells/metabolism
• Hematopoietic Stem Cells/cytology
• Humans
• Hyaluronan Receptors/metabolism
• Ischemia/metabolism
• Kidney/injuries
• Kidney/metabolism
• Macrophages/metabolism
• Mesenchymal Stem Cells/cytology
• Mice, Inbred C57BL

• adipose
• bone
• cells
• embryonic
• engineering
• marrow
• scaffold
• stem
• tissue
• umbilical

To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord contusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neurotrophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These findings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.

• adipose-derived stem cells
• cell transplantation
• glial cell line-derived neurotrophic factor
• motoneurons
• nerve regeneration
• neural regeneration
• neurotrophic factor
• spinal cord contusion injury
• spinal cord injury

Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

• bone marrow
• isolation
• mesenchymal stem cells
• mouse
• protocol

Since articular cartilage possesses only a weak capacity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is associated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechanical properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical procedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morphological features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells (MSCs) to be an appropriate cellular material for articular cartilage repair. These cells were originally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic differentiation is an inherent property of MSCs noticed at the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative properties. Moreover, these cells possess a considerable immunomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.

• Mesenchymal stem cells
• Regeneration
• Articular cartilage
• Cell therapy

• AFP
• Adipose tissue&ndash;derived mesenchymal stem cells
• CK18
• DMSO
• HNF4a
• HNF6
• glycogen storage
• hepatic differentiation

• Airway remodeling
• BALB/c
• Chronic model of asthma
• Mesenchymal stem cells
• Ovalbumin

Although preparation of early-stage bone-marrow-derived mesenchymal stem cells (BM-MSCs) is critical for successful cell transplantation therapy, no culture system offers a sufficient number of early-stage BM-MSCs for cell transplantation. Accordingly, we developed a culture system capable of producing a large number of early-stage BM-MSCs by using gelatin-coated matrix. The greatest retrieval and proliferation rates of the earliest-stage rat BM-MSCs were detected in bone-marrow-derived cells cultured on 1% (wt/v) gelatin-coated matrix, which showed significantly greater colony forming unit-fibroblast number, diameter, and total cell number. Moreover, continuous culture of the earliest-stage BM-MSCs on 1% (wt/v) gelatin-coated matrix resulted in a maximum of 21.2 ± 2.7 fold increase in the cumulative total number of early-stage BM-MSCs at passage 5. BM-MSCs generated in large quantities due to a reduced doubling time and an increased yield of cell population in S/G2/M phase showed typical fibroblast-like morphology and no significant differences in BM-MSC-related surface marker expression and differentiation potential, except for an increased ratio of differentiation into a neurogenic lineage. The use of gelatin-coated matrix in the retrieval and culture of BM-MSCs contributes greatly to the effective isolation and mass production of early-stage BM-MSCs.

• Bone Marrow Cells/cytology
• Bone Marrow Cells/drug effects
• Cartilage/cytology
• Cell Differentiation/drug effects
• Cells, Cultured
• Chondrocytes/cytology
• Chondrocytes/drug effects
• Dose-Response Relationship, Drug
• Glycogen Synthase Kinase 3/antagonists & inhibitors
• Glycogen Synthase Kinase 3/metabolism
• Humans
• Indoles/pharmacology
• Lithium Chloride/pharmacology
• Maleimides/pharmacology
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/drug effects
• Structure-Activity Relationship

• Balb/c strain
• CBA/Ca strain
• Differentiation
• ICR strain
• Immunophenotyping
• Mesenchymal stem cells
• Mouse bone marrow

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.

• Adipogenesis/physiology
• Animals
• Cell Differentiation/physiology
• Cell Separation/methods
• Chondrogenesis/physiology
• Female
• Immunophenotyping
• Knee Joint/anatomy & histology
• Knee Joint/cytology
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/metabolism
• Mice
• Mice, Inbred BALB C
• Osteogenesis/physiology
• Primary Cell Culture
• Synovial Membrane/cytology

While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs.

In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments.

These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

• Animals
• Anti-Inflammatory Agents/metabolism
• Biomarkers/metabolism
• Bone Marrow Cells/cytology
• Bone Marrow Cells/drug effects
• Bone Marrow Cells/metabolism
• Cell Differentiation/drug effects
• Cell Proliferation/drug effects
• Cell Separation/methods
• Cells, Cultured
• Cellular Senescence/drug effects
• Epiphyses/cytology
• Extremities/blood supply
• Fracture Healing/drug effects
• Fractures, Bone/pathology
• Fractures, Bone/therapy
• Immune Tolerance/drug effects
• Immunomodulation/drug effects
• Immunophenotyping
• Interferon-gamma/pharmacology
• Ischemia/pathology
• Ischemia/therapy
• Mesenchymal Stem Cell Transplantation
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/drug effects
• Mesenchymal Stem Cells/immunology
• Mesenchymal Stem Cells/metabolism
• Mice
• Models, Biological
• Multipotent Stem Cells/cytology
• Multipotent Stem Cells/drug effects
• Necrosis

The aim of this study was to determine whether the inflammatory milieu and/or hypoxia induces the dedifferentiation of synovial cells into mesenchymal stem-like cells, which may contribute to the tumor-like growth of synovial cells. Expression of mesenchymal stem cell markers (CD24, CD44, CD90, CD106, CD146 and Stro-1) was compared among cultured fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), bone marrow mesenchymal stem cells (BM MSCs) and normal dermal fibroblasts. After the cells were stimulated with pro-inflammatory cytokines for 3 days under hypoxia or normoxia, the stem cell markers were analyzed by FACS. CD44 and CD90 were expressed constitutively in all four cell types. Only the BM MSCs strongly expressed CD146. The expression of stem cell markers was similar between FLSs from RA and those from OA patients. In addition, the expression levels in FLSs were similar to those in normal dermal fibroblasts. The stimulation of FLSs and dermal fibroblasts with IL-1β or a mixture of cytokines under hypoxia did not induce a marked change in the expression of stem cell markers. These results indirectly suggest that the pro-inflammatory milieu may be not sufficient to induce the dedifferentiation of FLSs in arthritic joints.